Method of ameliorating the harmful effects of TNF using a polypeptide having the ability to bind to TNF

ABSTRACT

The present invention provides DNA sequences coding for a TNF-binding protein and for the TNF receptor of which this protein constitutes the soluble domain. The DNA sequences can be used for preparing recombinant DNA molecules in order to produce TNF-binding protein and TNF receptor. Recombinant TNF-binding protein is used in pharmaceutical preparations for treating indications in which TNF has a harmful effect. With the aid of the TNF receptor or fragments thereof or with the aid of suitable host organisms transformed with recombinant DNA molecules containing the DNA which codes for the TNF receptor or fragments or modifications thereof, it is possible to investigate substances for their interaction with the TNF receptor and/or for their effect on the biological activity of TNF.

This application is a division of U.S. application Ser. No. 09/525,998,filed Mar. 15, 2000, which is a continuation of U.S. application Ser.No. 08/383,676, filed Feb. 1, 1995, now U.S. Pat. No. 6,294,352, whichis a continuation of U.S. application Ser. No. 08/153,287, filed Nov.17, 1993, now abandoned, which is a continuation of U.S. applicationSer. No. 07/821,750, filed Jan. 1, 1992, now abandoned, which is adivision of U.S. application Ser. No. 07/511,430, filed Apr. 20, 1990,now abandoned, the contents of each of which are incorporated herein byreference. This application also claims priority to foreign patentapplications P39 13 101.7, filed Apr. 21, 1989, in Germany; P39 20282.8, filed Jun. 21, 1989, in Germany; and 90106624.1, filed Apr. 20,1990 in Europe (EPO).

FIELD OF THE INVENTION

The invention is in the field of recombinant genetics. In particular,the invention relates to a TNF receptor and to a TNF binding proteinproduced by recombinant means.

BACKGROUND OF THE INVENTION

Tumour necrosis factor (TNF-α) was first found in the serum of mice andrabbits which had been infected with Bacillus Calmette-Guerin and whichhad been injected with endotoxin, and was recognized on the basis of itscytotoxic and antitumor properties (Carswell, E. A., et al., Proc. Natl.Acad. Sci. 25:3666–3670 (1975)). It is produced particularly byactivated macrophages and monocytes.

Numerous types of cells which are targets of TNF have surface receptorswith a high affinity for this polypeptide (Old, L. J., Nature326:330–331 (1987)); it was assumed that lymphotoxin (TNF-β) binds tothe same receptor (Aggarwal, B. B., et al., Nature 318:655–667 (1985);Gullberg, U., et al., Eur. J. Haematol. 39:241–251 (1987)). TNF-α isidentical to a factor referred to as cachectin (Beutler, B., et al.,Nature 316:552–554 (1985)) which suppresses lipoprotein lipase andresults in hypertriglyceridaemia in chronically inflammatory andmalignant diseases (Torti, F. M. et al., Nature 229:867–869 (1985);Mahoney, J. R., et al., J. Immunol. 134:1673–1675 (1985)). TNF-α wouldappear to be involved in growth regulation and in the differentiationand function of cells which are involved in inflammation, immuneprocesses, and hematopoieses.

TNF can have a positive effect on the host organism by stimulatingneutrophils (Shalaby, M. R., et al., J. Immunol. 135:2069–2073 (1985);Klebanoff, S. J., et al., J. Immunol. 136:4220–4225 (1986)) andmonocytes and by inhibiting the replication of viruses (Mestan, J., etal., Nature 323:816–819 (1986); Wong, G. H. W., et al., Nature323:819–822 (1986)). Moreover, TNF-α activates the immune defensesagainst parasites and acts directly and/or indirectly as a mediator inimmune reactions, inflammatory processes, and other processes in thebody, although the mechanisms by which it works have not yet beenclarified in a number of cases. However, the administration of TNF-α(Cerami, A., et al., Immunol. Today 9:28–31 (1988)) can also beaccompanied by harmful phenomena (Tracey, K. J., et al., Science234:470–474 (1986)) such as shock and tissue damage, which can beremedied by means of antibodies against TNF-α (Tracey, K. J., et al.,Nature 330:662–666 (1987)).

A number of observations lead one to conclude that endogenously releasedTNF-α is involved in various pathological conditions. Thus, TNF-αappears to be a mediator of cachexia which can occur in chronicallyinvasive, e.g., parasitic, diseases. TNF-α also appears to play a majorpart in the pathogenesis of shock caused by gram negative bacteria(endotoxic shock); it would also appear to be implicated in some if notall the effects of lipopolysaccharides (Beutler B., et al., Ann. Rev.Biochem. 57:505–18 (1988)). TNF has also been postulated to have afunction in the tissue damage which occurs in inflammatory processes inthe joints and other tissues, and in the lethality and morbidity of thegraft-versus-host reaction (GVHR, Transplant Rejection (Piguet, P. F.,et al., Immunobiol. 175:27 (1987)). A correlation has also been reportedbetween the concentration of TNF in the serum and the fatal outcome ofmeningococcal diseases (Waage, A., et al., Lancet 1:355–357 (1987)).

It has also been observed that the administration of TNF-α over alengthy period causes a state of anorexia and malnutrition which hassymptoms similar to those of cachexia, which accompany neoplastic andchronic infectious diseases (Oliff A., et al., Cell 555–63 (1987)).

It has been reported that a protein derived from the urine of feverpatients has a TNF inhibiting activity; the effect of this protein ispresumed to be due to a competitive mechanism at the level of thereceptors (similar to the effect of the interleukin-1 inhibitor(Seckinger, P., et al., J. Immunol. 139:1546–1549 (1987); Seckinger P.,et al., J. Exp. Med., 1511–16 (1988)).

EP-A2 308 378 describes a TNF inhibiting protein obtained from humanurine. Its activity was demonstrated in the urine of healthy and illsubjects and determined on the basis of its ability to inhibit thebinding of TNF-α to its receptors on human HeLa cells and FS 11fibroblasts and the cytotoxic effect of TNF-α on murine A9 cells. Theprotein was purified until it became substantially homogeneous andcharacterized by its N-terminus. This patent publication does indeedoutline some theoretically possible methods of obtaining the DNA codingfor the protein and the recombinant protein itself; however, there is noconcrete information as to which of the theoretically possible solutionsis successful.

SUMMARY OF THE INVENTION

The invention relates to DNA coding for a TNF receptor protein or afragment thereof. In particular, the invention relates to DNA coding forthe TNF receptor protein having the formula

ATG GGC CTC TCC ACC GTG CCT GAC CTG CTG CTG CCA CTG GTG CTC CTG GAG CTGTTG GTG GGA ATA TAC CCC TCA GGG GTT ATT GGA CTG GTC CCT CAC CTA GGG GACAGG GAG AAG AGA GAT AGT GTG TGT CCC CAA GGA AAA TAT ATC CAC CCT CAA AATAAT TCG ATT TGC TGT ACC AAG TGC CAC AAA GGA ACC TAC TTG TAC AAT GAC TGTCCA GGC CCG GGG CAG GAT ACG GAC TGC AGG GAG TGT GAG AGC GGC TCC TTC ACCGCT TCA GAA AAC CAC CTC AGA CAC TGC CTC AGC TGC TCC AAA TGC CGA AAG GAAATG GGT CAG GTG GAG ATC TCT TCT TGC ACA GTG GAC CGG GAC ACC GTG TGT GGCTGC AGG AAG AAC CAG TAC CGG CAT TAT TGG AGT GAA AAC CTT TTC CAG TGC TTCAAT TGC AGC CTC TGC CTC AAT GGG ACC GTG CAC CTC TCC TGC CAG GAG AAA CAGAAC ACC GTG TGC ACC TGC CAT GCA GGT TTC TTT CTA AGA GAA AAC GAG TGT GTCTCC TGT AGT AAC TGT AAG AAA AGC CTG GAG TGC ACG AAG TTG TGC CTA CCC CAGATT GAG AAT GTT AAG GGC ACT GAG GAC TCA GGC ACC ACA GTG CTG TTG CCC CTGGTC ATT TTC TTT GGT CTT TGC CTT TTA TCC CTC CTC TTC ATT GGT TTA ATG TATCGC TAC CAA CGG TGG AAG TCC AAG CTC TAC TCC ATT GTT TGT GGG AAA TCG ACACCT GAA AAA GAG GGG GAG CTT GAA GGA ACT ACT ACT AAG CCC CTG GCC CCA AACCCA AGC TTC AGT CCC ACT CCA GGC TTC ACC CCC ACC CTG GGC TTC AGT CCC GTGCCC AGT TCC ACC TTC ACC TCC AGC TCC ACC TAT ACC CCC GGT GAC TGT CCC AACTTT GCG GCT CCC CGC AGA GAG GTG GCA CCA CCC TAT CAG GGG GCT GAC CCC ATCCTT GCG ACA GCC CTC GCC TCC GAC CCC ATC CCC AAC CCC CTT CAG AAG TGG GAGGAC AGC GCC CAC AAG CCA CAG AGC CTA GAG ACT GAT GAC CCC GCG ACG CTG TACGCC GTG GTG GAG AAC GTG CCC CCG TTG CGC TGG AAG GAA TTC GTG CGG CGC CTAGGG CTG AGC GAC CAC GAG ATC GAT CGG CTG GAG CTG CAG AAC GGG CGC TGC CTGCGC GAG GCG CAA TAC AGC ATG CTG GCG ACC TGG AGG CGG CGC ACG CCG CGG CGCGAG GCC ACG CTG GAG CTG CTG GGA CGC GTG CTC CGC GAC ATG GAC CTG CTG GGCTGC CTG GAG GAG ATC GAG GAG GCG CTT TGC GGC CCC GCC GCC CTC CCG CCC GCGCCC AGT CTT CTC AGA TGAor a fragment or a degenerate variant thereof.

The invention also relates to DNA coding for a secretable TNF-bindingprotein having the formula

R²  GAT AGT GTG TGT CCC CAA GGA AAA TAT ATC CAC CCT CAA AAT AAT TCG ATTTGC TGT ACC AAG TGC CAC AAA GGA ACC TAC TTG TAC AAT GAC TGT CCA GGC CCGGGG CAG GAT ACG GAC TGC AGG GAG TGT GAG AGC GGC TCC TTC ACC GCT TCA GAAAAC CAC CTC AGA CAC TGC CTC AGC TGC TCC AAA TGC CGA AAG GAA ATG GGT CAGGTG GAG ATC TCT TCT TGC ACA GTG GAC CGG GAC ACC GTG TGT GGC TGC AGG AAGAAC CAG TAC CGG CAT TAT TGG AGT GAA AAC CTT TTC CAG TGC TTC AAT TGC AGCCTC TGC CTC AAT GGG ACC GTG CAC CTC TCC TGC CAG GAG AAA CAG AAC ACC GTGTGC ACC TGC CAT GCA GGT TTC TTT CTA AGA GAA AAC GAG TGT GTC TCC TGT AGTAAC TGT AAG AAA AGC CTG GAG TGC ACG AAG TTG TGC CTA CCC CAG ATT GAG AATwherein R² is optionally absent or represents DNA coding for apolypeptide which can be cleaved in vivo; or a degenerate variantthereof.

The invention also relates to nucleic acid which hybridizes with the DNAof the invention under conditions of low stringency and which codes fora polypeptide having the ability to bind TNF.

The invention also relates to a recombinant DNA molecule, comprising theDNA molecules of the invention.

The invention also relates to host cells transformed with therecombinant DNA molecules of the invention.

The invention also relates to the substantially pure recombinant TNFreceptor polypeptides of the invention. In particular, the inventionrelates to a TNF receptor of formula

met gly leu ser thr val pro asp leu leu leu pro leu val leu leu glu leuleu val gly ile tyr pro ser gly val ile gly leu val pro his leu gly asparg glu lys arg asp ser val cys pro gln gly lys tyr ile his pro gln asnasn ser ile cys cys thr lys cys his lys gly thr tyr leu tyr asn asp cyspro gly pro gly gln asp thr asp cys arg glu cys glu ser gly ser phe thrala ser glu asn his leu arg his cys leu ser cys ser lys cys arg lys glumet gly gln val glu ile ser ser cys thr val asp arg asp thr val cys glycys arg lys asn gln tyr arg his tyr trp ser glu asn leu phe gln cys pheasn cys ser leu cys leu asn gly thr val his leu ser cys gln glu lys glnasn thr val cys thr cys his ala gly phe phe leu arg glu asn glu cys valser cys ser asn cys lys lys ser leu glu cys thr lys leu cys leu pro glnile glu asn val lys gly thr glu asp ser gly thr thr val leu leu pro leuval ile phe phe gly leu cys leu leu ser leu leu phe ile gly leu met tyrarg tyr gln arg trp lys ser lys leu tyr ser ile val cys gly lys ser thrpro glu lys glu gly glu leu glu gly thr thr thr lys pro leu ala pro asnpro ser phe ser pro thr pro gly phe thr pro thr leu gly phe ser pro valpro ser ser thr phe thr ser ser ser thr tyr thr pro gly asp cys pro asnphe ala ala pro arg arg glu val ala pro pro tyr gln gly ala asp pro ileleu ala thr ala leu ala ser asp pro ile pro asn pro leu gln lys trp gluasp ser ala his lys pro gln ser leu asp thr asp asp pro ala thr leu tyrala val val glu asn val pro pro leu arg trp lys glu phe val arg arg leugly leu ser asp his glu ile asp arg leu glu leu gln asn gly arg cys leuarg glu ala gln tyr ser met leu ala thr trp arg arg arg thr pro arg argglu ala thr leu glu leu leu gly arg val leu arg asp met asp leu leu glycys leu glu asp ile glu glu ala leu cys gly pro ala ala leu pro pro alapro ser leu leu argor a fragment thereof which binds to TNF.

The invention also relates to the TNF binding protein of the formula

asp ser val cys pro gln gly lys tyr ile his pro gln asn asn ser ile cyscys thr lys cys his lys gly thr tyr leu tyr asn asp cys pro gly pro glygln asp thr asp cys arg glu cys glu ser gly ser phe thr ala ser glu asnhis leu arg his cys leu ser cys ser lys cys arg lys glu met gly gln valglu ile ser ser cys thr val asp arg asp thr val cys gly cys arg lys asngln tyr arg his tyr trp ser glu asn leu phe gln cys phe asn cys ser leucys leu asn gly thr val his leu ser cys gln glu lys gln asn thr val cysthr cys his ala gly phe phe leu arg glu asn glu cys val ser cys ser asncys lys lys ser leu glu cys thr lys leu cys leu pro gln ile glu asnor a functional derivative or fragment thereof having the ability tobind TNF.

The invention also relates to a process for preparing a recombinant TNFreceptor protein, or a functional derivative thereof which is capable ofbinding to TNF, comprising cultivating a host cell of the invention andisolating the expressed recombinant TNF receptor protein.

The invention also relates to pharmaceutical compositions comprising aTNF receptor protein, or a functional derivative or fragment thereof,and a pharmaceutically acceptable carrier.

The invention also relates to a method for ameliorating the harmfuleffects of TNF in an animal, comprising administering to an animal inneed of such treatment a therapeutically effective amount of a TNFreceptor polypeptide, or fragment thereof which binds to TNF.

The invention also relates to a method for the detection of TNF in abiological sample, comprising contacting said sample with an effectiveamount of a TNF receptor polypeptide, or fragment thereof which binds toTNF, and detecting whether a complex is formed.

DESCRIPTION OF THE FIGURES

FIGS. 1A–1C depict the complete nucleotide sequence (SEQ ID NO: 21) of1334 bases of the cDNA insert of λ-TNF-BP15 and pTNF-BP15.

FIG. 2 depicts a hydrophobicity profile which was produced using the MacMolly program.

FIGS. 3A–3B depict the scheme used for the construction of plasmidpCMV-SV40.

FIGS. 4A–4B depict the scheme used for the construction of plasmidpSV2gptDHFR Mut2.

FIGS. 5A–5B depict the scheme used for the construction of plasmidspAD-CMV1 and pAD-CMV2.

FIGS. 6A–6E depict the full nucleotide sequence (SEQ ID NO: 23) of the6414 bp plasmid pAD-CMV1.

FIGS. 7A–7D depicts the structure of the plasmids designated pADTNF-BP,pADBTNF-BP, pADTNF-R, and pADBTNF-R.

FIGS. 8A–8B depict the complete nucleotide sequence (SEQ ID NO: 24) ofraTNF-R8.

FIGS. 9A–9B depict the complete coding region (SEQ ID NO: 26) for humanTNF-R in lTNF-R2.

FIG. 10 depicts an autoradiogram showing a singular RNA band with alength of 2.3 kb for the human TNF receptor.

DETAILED DESCRIPTION OF THE INVENTION

In preliminary tests for the purposes of the present invention (seeExamples 1–4), a protein was identified from the dialyzed urine ofuraemia patients, and this protein inhibits the biological effects ofTNF-α by interacting with TNF-α to prevent it from binding to its cellsurface receptor (Olsson I., et al., Eur. J. Haematol. 41:414–420(1988)). This protein was also found to have an affinity for TNF-β.

The presence of this protein (hereinafter referred to as TNF-BP) inconcentrated dialyzed urine was detected by competition with the bindingof radioactively labeled recombinant TNF-α to a subclone of HL-60 cells,by measuring the influence of dialyzed urine on the binding of¹²⁵I-TNF-α to the cells. The binding tests carried out showed a dosagedependent inhibition of TNF-α binding to the cells by concentrateddialyzed urine (the possible interpretation that the reduction inbinding observed might be caused by any TNF-α present in the urine or byTNF-β competing for the binding, was ruled out by the discovery that thereduction in binding could not be remedied by the use of TNF-α and TNF-βantibodies).

Analogously, in preliminary tests for the purposes of the presentinvention, it was demonstrated that TNF-BP also shows an affinity forTNF-β, which is about 1/50 of its affinity for TNF-α.

Gel chromatography on Sephacryl 200 showed that a substance in the urineand serum of dialysis patients and in the serum of healthy subjectsforms a complex with recombinant TNF-α with a molecular weight of about75,000.

TNF-BP was concentrated 62 times from several samples of dialyzed urinefrom uraemia patients by partial purification using pressureultrafiltration, ion exchange chromatography, and gel chromatography.

The preparations obtained were used to detect the biological activity ofTNF-BP by inhibiting the growth-inhibiting effect of TNF-α on HL-60-10cells. TNF-BP was found to have a dosage dependent effect on thebiological activity of TNF-α. The binding characteristics of cells werealso investigated by pretreatment with TNF-BP and an exclusivecompetition binding test. It was shown that pretreatment of the cellswith TNF-BP does not affect the binding of TNF-α to the cells. Thisindicates that the effect of TNF-BP is not based on any binding to thecells and competition with TNF-α for the binding to the receptor.

The substantially homogeneous protein is obtained in highly purifiedform by concentrating urine from dialysis patients by ultrafiltration,dialyzing the concentrated urine, and concentrating it four-fold in afirst purification step using DEAE sephacel chromatography. Furtherconcentration was carried out by affinity chromatography usingsepharose-bound TNF-α. The final purification was carried out usingreverse phase chromatography (FPLC).

It was shown that the substantially highly purified protein inhibits thecytotoxic effect of TNF-α on WEHI 164 clone 13 cells (Olsson et al.,Eur. J. Haematol. 42:270–275 (1989)).

The N-terminal amino acid sequence of the substantially highly purifiedprotein was analyzed. It was found to beAsp-Ser-Val-Xaa-Pro-Gln-Gly-Lys-Tyr-Ile-His-Pro-Gln (main sequence; SEQID NO: 28); the following N-terminal sequence was detected in traces:Leu-(Val)-(Pro)-(His)-Leu-Gly-Xaa-Arg-Glu (subsidiary sequence; SEQ IDNO: 29). A comparison of the main sequence with the Nterminal sequenceof the TNF-inhibiting protein disclosed in EP-A2 308 378 shows that thetwo proteins are identical.

The following amino acid composition was found, given in mols of aminoacid per mol of protein and in mol % of amino acid, measured as theaverage of 24-hour and 48-hour hydrolysis:

Mol of amino acid/ Mol % amino mol of protein acid Asp + Asn 27.5 10.9Thr 15.8 6.3 Ser 20.7 8.2 Glu + Gln 35.0 13.8 Pro 9.5 3.8 Gly 16.0 6.3Ala 4.2 1.7 Cys 32.3 12.8 Val 10.8 4.3 Met 1.1 0.4 Ile 7.0 2.8 Leu 20.28.0 Tyr 6.1 2.4 Phe 8.1 3.2 His 11.1 4.4 Lys 15.7 6.2 Arg 11.8 4.7 Total252.9 100

A content of glucosamine was detected by amino acid analysis. Theresults of an affinoblot carried out using Concanavalin A and wheatgermlectin also showed that TNF-BP is a glycoprotein.

The substantially homogeneous protein was digested with trypsin and theamino acid sequences of 17 of the cleavage peptides obtained weredetermined. The C-terminus was also analyzed.

TNF-BP obviously has the function of a regulator of TNF activity withthe ability to buffer the variations in concentration of free,biologically active TNF-α. TNF-BP should also affect the secretion ofTNF by the kidneys because the complex formed with TNF, the molecularweight of which was measured at around 75,000 by gel permeationchromatography on Sephadex G 75, is obviously not retained by theglomerulus, unlike TNF. The TNF-BP was detected in the urine of dialysispatients as one of three main protein components which have an affinityfor TNF and which are eluted together with TNF-BP from the TNF affinitychromatography column. However, the other two proteins obviously bind ina manner which does not affect the binding of TNF-α to its cell surfacereceptor.

The results obtained regarding the biological activity of TNF-BP, inparticular the comparison of the binding constant with the bindingconstant described for the TNF receptor (Creasey, A. A., et al., Proc.Natl. Acad. Sci. 84:3293–3297 (1987)), provided a first indication thatthis protein might be the soluble part of a TNF receptor.

In view of its ability to inhibit the biological activity of TNF-α andTNF-β, the TNF binding protein is suitable for use in cases where areduction in the TNF activity in the body is indicated. Functionalderivatives or fragments of the TNF binding protein with the ability toinhibit the biological activity of TNF are also suitable for use in suchcases.

Covalent modifications of the TNF binding proteins of the presentinvention are included within the scope of this invention. Variant TNFbinding proteins may be conveniently prepared by in vitro synthesis.Such modifications may be introduced into the molecule by reactingtargeted amino acid residues of the purified or crude protein with anorganic derivatizing agent that is capable of reacting with selectedside chains or terminal residues. The resulting covalent derivatives areuseful in programs directed at identifying residues important forbiological activity.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonateat pH 5.5–7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylissurea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues per se has been studiedextensively, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizol and tetranitromethaneare used to form O-acetyl tyrosyl species and 3-nitro derivatives,respectively. Tyrosyl residues are iodinated using ¹²⁵I or ¹³¹I toprepare labeled proteins for use in radioimmunoassay, the chloramine Tmethod described above being suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′-N—C—N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3 (4azonia 4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl andglutamyl residues are converted to asparaginyl and glutaminyl residuesby reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking theTNF binding proteins to water-insoluble support matrixes or surfaces foruse in the method for cleaving TNF binding protein-fusion polypeptide torelease and recover the cleaved polypeptide. Commonly used crosslinkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or theonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MoleculeProperties, W.H. Freeman & Co., San Francisco, pp. 79–86 (1983)),acetylation of the N-terminal amine, and, in some instances, amidationof the C-terminal carboxyl groups.

Amino acid sequence variants of the TNF binding proteins can also beprepared by mutations in the DNA. Such variants include, for example,deletions from, or insertions or substitutions of, residues within theamino acid sequence shown in the Figures. Any combination of deletion,insertion, and substitution may also be made to arrive at the finalconstruct, provided that the final construct possesses the desiredactivity (binding to TNF). Obviously, the mutations that will be made inthe DNA encoding the variant must not place the sequence out of readingframe and preferably will not create complementary regions that couldproduce secondary mRNA structure (see EP Patent Application PublicationNo. 75,444).

At the genetic level, these variants ordinarily are prepared bysite-directed mutagenesis of nucleotides in the DNA encoding the TNFbinding proteins, thereby producing DNA encoding the variant, andthereafter expressing the DNA in recombinant cell culture. The variantstypically exhibit the same qualitative biological activity as thenaturally occurring analog.

While the site for introducing an amino acid sequence variation ispredetermined, the mutation per se need not be predetermined. Forexample, to optimize the performance of a mutation at a given site,random mutagenesis may be conducted at the target codon or region andthe expressed TNF binding protein variants screened for the optimalcombination of desired activity. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown, for example, site-specific mutagenesis.

Preparation of a TNF binding protein variant in accordance herewith ispreferably achieved by site-specific mutagenesis of DNA that encodes anearlier prepared variant or a nonvariant version of the protein.Site-specific mutagenesis allows the production of TNF binding proteinvariants through the use of specific oligonucleotide sequences thatencode the DNA sequence of the desired mutation, as well as a sufficientnumber of adjacent nucleotides, to provide a primer sequence ofsufficient size and sequence complexity to form a stable duplex on bothsides of the deletion junction being traversed. Typically, a primer ofabout 20 to 25 nucleotides in length is preferred, with about 5 to 10residues on both sides of the junction of the sequence being altered. Ingeneral, the technique of site-specific mutagenesis is well known in theart, as exemplified by publications such as Adelman et al., DNA 2:183(1983), the disclosure of which is incorporated herein by reference.

As will be appreciated, the site-specific mutagenesis techniquetypically employs a phage vector that exists in both a single-strandedand double-stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage, for example, asdisclosed by Messing et al., Third Cleveland Symposium on Macromoleculesand Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981), thedisclosure of which is incorporated herein by reference. These phage arereadily commercially available and their use is generally well known tothose skilled in the art. Alternatively, plasmid vectors that contain asingle-stranded phage origin of replication (Veira et al., Meth.Enzymol. 153:3 (1987)) may be employed to obtain single-stranded DNA.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector that includeswithin its sequence a DNA sequence that encodes the relevant protein. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically, for example, by the method of Crea et al.,Proc. Natl. Acad. Sci. (USA) 75:5765 (1978). This primer is thenannealed with the single-stranded protein-sequence-containing vector,and subjected to DNA-polymerizing enzymes such as E. coli polymerase IKlenow fragment, to complete the synthesis of the mutation-bearingstrand. Thus, a mutated sequence and the second strand bear the desiredmutation. This heteroduplex vector is then used to transform appropriatecells such as JM101 cells, and clones are selected that includerecombinant vectors bearing the mutated sequence arrangement.

After such a clone is selected, the mutated protein region may beremoved and placed in an appropriate vector for protein production,generally an expression vector of the type that may be employed fortransformation of an appropriate host.

Amino acid sequence deletions may generally range from about 1 to 30residues or 1 to 10 residues, and typically are contiguous.

Amino acid sequence insertions include amino and/or carboxyl-terminalfusions of from one residue to polypeptides of essentially unrestrictedlength, as well as intrasequence insertions of single or multiple aminoacid residues. Intrasequence insertions (i.e., insertions within thecomplete hormone receptor molecule sequence) may range generally fromabout 1 to 10 or 1 to 5 residues. An example of a terminal insertionincludes a fusion of a signal sequence, whether heterologous orhomologous to the host cell, to the N-terminus of the TNF bindingprotein to facilitate the secretion of mature TNF binding protein fromrecombinant hosts.

The third group of variants are those in which at least one amino acidresidue in the TNF binding protein, and preferably, only one, has beenremoved and a different residue inserted in its place. Suchsubstitutions preferably are made in accordance with the following Table1 when it is desired to modulate finely the characteristics of a hormonereceptor molecule.

TABLE 1 Exemplary Original Residue Substitutions Ala gly; ser Arg lysAsn gln; his Asp glu Cys ser Gln asn Glu asp Gly ala; pro His asn; glnIle leu; val Leu ile; val Lys arg; gln; glu Met leu; tyr; ile Phe met;leu; tyr Ser thr Thr ser Trp tyr Tyr trp; phe Val ile; leu

Substantial changes in functional or immunological identity are made byselecting substitutions that are less conservative than those in Table1, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain. The substitutions thatin general are expected to those in which (a) glycine and/or proline issubstituted by another amino acid or is deleted or inserted; (b) ahydrophilic residue, e.g., seryl or threonyl, is substituted for (or by)a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, oralanyl; (c) a cysteine residue is substituted for (or by) any otherresidue; (d) a residue having an electropositive side chain, e.g.,lysyl, arginyl, or histidyl, is substituted for (or by) a residue havingan electronegative charge, e.g., glutamyl or aspartyl; or (e) a residuehaving a bulky side chain, e.g., phenylalanine, is substituted for (orby) one not having such a side chain, e.g., glycine.

Most deletions and insertions, and substitutions in particular, are notexpected to produce radical changes in the characteristics of the TNFbinding protein. However, when it is difficult to predict the exacteffect of the substitution, deletion, or insertion in advance of doingso, one skilled in the art will appreciate that the effect will beevaluated by routine screening assays. For example, a variant typicallyis made by site-specific mutagenesis of the TNF binding protein-encodingnucleic acid, expression of the variant nucleic acid in recombinant cellculture, and, optionally, purification from the cell culture, forexample, by immunoaffinity adsorption on a polyclonal anti-TNF bindingprotein column (to absorb the variant by binding it to at least oneremaining immune epitope).

The activity of the cell lysate or purified TNF binding protein variantis then screened in a suitable screening assay for the desiredcharacteristic. For example, a change in the binding affinity for TNF orimmunological character of the TNF binding protein, such as affinity fora given antibody, is measured by a competitive type immunoassay. Changesin immunomodulation activity are measured by the appropriate assay.Modifications of such protein properties as redox or thermal stability,hydrophobicity, susceptibility to proteolytic degradation or thetendency to aggregate with carriers or into multimers are assayed bymethods well known to the ordinarily skilled artisan.

TNF-BP (or the functional derivatives, variants, and active fragmentsthereof) may be used for the prophylactic and therapeutic treatment ofthe human or animal body in indications where TNF-α has a harmfuleffect. Such diseases include in particular inflammatory and infectiousand parasitic diseases or states of shock in which endogenous TNF-α isreleased, as well as cachexia, GVHR, ARDS (Adult Respiratory DistressSymptom) and autoimmune diseases such as rheumatoid arthritis, etc. Alsoincluded are pathological conditions which may occur as side effects oftreatment with TNF-α, particularly at high doses, such as severehypotension or disorders of the central nervous system.

In view of its TNF binding properties, TNF-BP is also suitable as adiagnostic agent for determining TNF-α and/or TNF-β, e.g., as one of thecomponents in radioimmunoassays or enzyme immunoassays, optionallytogether with antibodies against TNF.

In view of its properties, this protein is a pharmacologically usefulactive substance which cannot be obtained in sufficient quantities fromnatural sources using protein-chemical methods.

There was therefore a need to produce this protein (or related proteinswith the ability to bind TNF) by recombinant methods in order that itcould be made available in sufficient amounts for therapeutic use. Thephrase “ability to bind TNF” within the scope of the present inventionmeans the ability of a protein to bind to TNF-α in such a way that TNF-αis prevented from binding to the functional part of the receptor and theactivity of TNF-α in humans or animals is inhibited or preventedaltogether. This definition also includes the ability of a protein tobind to other proteins, e.g., to TNF-β, and inhibit their effect.

The aim of the present invention was to provide the DNA which codes forTNF-BP, in order to make it possible, on the basis of this DNA, toproduce recombinant DNA molecules by means of which suitable hostorganisms can be transformed, with the intention of producing TNF-BP orfunctional derivatives and fragments thereof.

Within the scope of this objective, it was also necessary to establishwhether TNF-BP is the soluble part of a TNF receptor. This assumptionwas confirmed, thus providing the basis for clarification of thereceptor sequence.

Another objective within the scope of the present invention was toprepare the cDNA coding for a TNF receptor, for the purpose of producingrecombinant human TNF receptor.

The presence of a specific receptor with a high affinity for TNF-α onvarious cell types was shown by a number of working groups. Recently,the isolation and preliminary characterization of a TNF-α receptor wasreported for the first time (Stauber, G. B., et al., J. Biol. Chem.263:19098–19104 (1988)). Since the binding of radioactively labeledTNF-α can be reversed by an excess of TNF-β (Aggarwal, B. B., et al.,Nature 318:655–667 (1985)), it was proposed that TNF-α and TNF-β share acommon receptor. On the other hand, since it was shown that certain celltypes which respond to TNF-α are wholly or partly insensitive to TNF-β(Locksley, R. M., et al., J. Imunol. 139:1891–1895 (1987)), theexistence of a common receptor was thrown into doubt again.

By contrast, recent results on the binding properties of TNF-β toreceptors appear to confirm the theory of a common receptor (Stauber, G.B., et al., J. Biol. Chem. 264:3573–3576 (1989)), and this studyproposes that there are differences between TNF-α and TNF-β in theirinteraction with the receptor or in addition with respect to the eventswhich occur in the cell after the ligand-receptor interaction. Lately,there has been a report of another TNF-binding protein which is presumedto be the soluble form of a different TNF receptor (Engelmann et al., J.Biol. Chem. 265:1531–1536 (1990)). The availability of the DNA codingfor a TNF receptor is the prerequisite for the production of recombinantreceptor and consequently makes it much easier to carry out comparativeinvestigations on different types of cell regarding their TNF-α and/orTNF-β receptors or the reactions triggered by the binding of TNF to thereceptor in the cell. It also makes it possible to clarify thethree-dimensional structure of the receptor and hence provide theprerequisite for a rational design for the development of agonists andantagonists for the TNF activity.

The efforts to solve the problem of the invention started from thefinding that major difficulties are occasionally encountered whensearching through cDNA libraries using hybridizing probes derived fromamino acid sequences of short peptides, on account of the degenerationof the genetic code. In addition, this procedure is made more difficultwhen the researcher does not know in which tissue a particular protein,e.g., TNF-BP, is synthesized. In this case, should the method fail, itis not always possible to tell with any certainty whether the cause ofthe failure was the choice of an unsuitable cDNA library or theinsufficient specificity of the hybridization probes.

Therefore, the following procedure was used according to the inventionin order to obtain the DNA coding for TNF-BP:

The cDNA library used was a library of the fibrosarcoma cell line HS913T which had been induced with TNF-α and was present in λ gt11. In orderto obtain λ DNA with TNF-BP sequences from this library, the high degreeof sensitivity of the polymerase chain reaction (PCR; Saiki, R. X.,Science 239:487–491 (1988)) was used. Using this method it is possibleto obtain, from an entire cDNA library, an unknown DNA sequence flankedby oligonucleotides which have been designed on the basis of known aminoacid partial sequences and used as primers. A longer DNA fragment ofthis kind can subsequently be used as a hybridization probe, e.g., inorder to isolate cDNA clones, particularly the original cDNA clone.

On the basis of the N-terminal amino acid sequence (main sequence) andamino acid sequences of tryptic peptides obtained from highly purifiedTNF-BP, hybridization probes were prepared. Using these probes, a cDNAwhich constitutes part of the cDNA coding for TNF-BP was obtained by PCRfrom the cDNA library HS913T.

This cDNA has the following nucleotide sequence:

CAG GGG AAA TAT ATT CAC CCT CAA AAT AAT TCG ATT TGC TGT ACC AAG TGC CACAAA GGA ACC TAC TTG TAC AAT GAC TGT CCA GGC CCG GGG CAG GAT ACG GAC TGCAGG GAG TGT GAG AGC GGC TCC TTC ACA GCC TCA GAA AAC AAC AAG

This DNA is one of a number of possible variants which are suitable forhybridizing with TNF-BP DNAs or TNF-BP-RNAs (these variants include, forexample, those DNA molecules which are obtained by PCR amplificationwith the aid of primers, wherein the nucleotide sequence does notcoincide precisely with the desired sequence, possibly as a result ofrestriction sites provided for cloning purposes or because of aminoacids which were not clearly identified in the amino acid sequenceanalysis). “TNF-BP-DNAs” and “TNF-BP-RNAs” indicate nucleic acids whichcode for TNF-BP or related proteins with the ability to bind TNF orwhich contain a sequence coding for such a protein.

TNF-BP-DNAs (or TNF-BP-RNAs) also include cDNAs derived from mRNAs whichare formed by alternative splicing (or these mRNAs themselves). Thephrase “alternative splicing” means the removal of introns, using spliceacceptor and/or splice donor sites which are different from the samemRNA precursor. The mRNAs thus formed differ from one another by thetotal or partial presence or absence of certain exon sequences, and insome cases there may be a shift in the reading frame.

The cDNA (or variants thereof) initially obtained according to theinvention, containing some of the sequence coding for TNF-BP, can thusbe used as a hybridization probe in order to obtain cDNA clonescontaining TNF-BP DNAs from cDNA libraries. It may also be used as ahybridization probe for mRNA preparations, for isolating TNF-BP RNAs andfor producing concentrated cDNA libraries therefrom, for example, toallow much simpler and more efficient screening. A further field ofapplication is the isolation of the desired DNAs from genomic DNAlibraries using these DNAs as hybridization probes.

The DNA defined hereinbefore (or a variant thereof) is capable ofhybridizing with DNAs (or RNAs) which code for TNF-BP or contain thesequence which codes for TNF-BP. Using this DNA as a probe, it is alsopossible to obtain cDNAs which code for proteins, the processing ofwhich yields TNF-BP. The term “processing” means the splitting off ofpartial sequences in vivo. This might mean, at the N-terminus the signalsequence and/or other sequences and possibly also at the C-terminus, thetransmembrane and cytoplasmic region of the receptor. Using thishybridization signal it is therefore possible to search through suitablecDNA libraries to look for any cDNA which contains the entire sequencecoding for a TNF receptor (if necessary, this operation may be carriedout in several steps).

According to the invention, the cDNA of the sequence definedhereinbefore, which had been obtained by PCR from the cDNA library ofthe TNF-α induced fibrosarcoma cell line HS913 T (in λ gt11), was usedto search through the cDNA library once more, the lambda DNA was excisedfrom the hybridizing clones, subcloned, and sequenced. A 1334 base longcDNA insert was obtained which contains the sequence coding for TNF-BP.

Thus, first of all, DNAs were prepared, coding for a polypeptide capableof binding TNF, or for a polypeptide in which this TNF binding proteinis a partial sequence. These DNAs also include DNAs of the kind whichcode for parts of these polypeptides.

The complete nucleotide sequence of the longest cDNA insert obtained isshown in FIGS. 1A–1C.

This nucleotide sequence has a continuous open reading frame beginningwith base 213 up to the end of the 1334 bp long cDNA insert. Since thereis a stop codon (TAG) in the same reading frame 4 codons before thepotential translation start codon ATG (213–215), it was assumed that thestart codon is actually the start of translation used in vivo.

A comparison of the amino acid sequence derived from the nucleotidesequence with the amino acid sequences determined from the aminoterminal end of TNF-BP and tryptic peptides, shows a high degree ofconformity. This means that the isolated cDNA contains the sequencecoding for authentic TNF-BP.

Starting from the N-terminus, the first sequence which shows conformitywith a tryptic cleavage peptide sequence is the sequence from fraction12 (Leu-Val-Pro- . . . ), which had also been obtained as a subsidiarysequence in the analysis of the N-terminus of TNF-BP. This N-terminalleucine corresponds to the 30th amino acid in the cDNA sequence. Sincethe preceding section of 29 amino acids has a strongly hydrophobicnature and TNF-BP is a secreted protein, it can be concluded that these29 amino acids constitute the signal peptide required for the secretionprocess, which is split off during secretion (designated S1–S29 in FIG.1A). The amino acid sequence obtained as the main sequence in theN-terminal analysis of TNF-BP corresponds to the amino acids beginningwith Asp-12 in the cDNA sequence. This aspartic acid group directlyfollows the basic dipeptide Lys-Arg. Since a very large number ofproteins are cleaved proteolytically in vivo after this dipeptide, itcan be assumed that TNF-BP with N-terminal Asp is not formed directly bythe processing of a precursor in the secretion process, but that theN-terminal 11 amino acids are split off from the processed protein at alater time by extracellular proteases. The carboxy-terminal end ofTNF-BP had been determined as Ile-Glu-Asn (C-terminal analysis; trypticpeptide fraction 27: amino acids 159–172, tryptic peptide fraction 21:amino acids 165–172), Asn corresponding to position 172 in the cDNAsequence.

Potential N-glycosylation sites of general formula Asn-Xaa-Ser/Thr, inwhich Xaa may be any amino acid other than proline, are located atpositions 25–27 (Asn-Asn-Ser), 116–118 (Asn-Cys-Ser), and 122–124(Asn-Gly-Thr) of the TNF-BP cDNA sequence. The fact that Asn-25 isglycosylated is clear from the fact that Asn could not be identified inthe sequencing of the corresponding tryptic cleavage peptide at thissite.

Analysis of the nucleotide sequence or the amino acid sequence derivedtherefrom in conjunction with the protein-chemical investigationscarried out shows that TNF-BP is a glycosylated polypeptide with 172amino acids, which is converted by proteolytic cleaving after the 11thamino acid into a glycoprotein with 161 amino acids. The following Tableshows the tryptic peptides sequenced and the corresponding amino acidsequences derived from the cDNA sequence:

Fraction Amino acids 12 1–8  1 12–19  8 20–32 14/I 36–48 20 36–53 1154–67 (Amino acids 66–67 had not been correctly determined on thepeptide) 14/lI 79–91 26 133–146  5 147–158 27 159–172

The cDNA obtained is the prerequisite for the preparation of recombinantTNF-BP.

As already mentioned, the cDNA initially isolated according to theinvention does not contain the stop codon which could have been expectedfrom analysis of the C-terminus after the codon for Asn-172, but theopen reading frame is continued. The region between Val-183 and Met-204is strongly hydrophobic by nature. This hydrophobic region of 22 aminoacids followed by a portion containing positively charged amino acids(Arg-206, Arg-209) has the typical features of a transmembrane domainwhich anchors proteins in the cell membrane. The protein fractionfollowing in the C-terminus direction, on the other hand, is stronglyhydrophilic.

The hydrophobicity profile is shown in FIG. 2 (the hydrophobicity plotwas produced using the MacMolly program; Soft Gene Berlin); the windowsize for calculating the values was 11 amino acids. Hydrophobic regionscorrespond to positive values and hydrophilic regions to negative valueson the ordinates. The abscissa shows the number of amino acids beginningwith the start methionine S1).

The protein structure shows that the DNA coding for the soluble TNF-BPsecreted is part of a DNA coding for a larger protein; this protein hasthe feature of a protein anchored in the cell membrane, contains TNF-BPin a manner typical of extracellular domains and has a substantialportion which is typical of cytoplasmatic domains. Soluble TNF-BP isobviously obtained from this membrane-bound form by proteolytic cleavingjust outside the transmembrane domain.

The structure of the protein coded by the cDNA obtained in conjunctionwith the ability of TNF-BP to bind TNF confirms the assumption thatTNF-BP is part of a cellular surface receptor for TNF the extracellulardomains of which, responsible for the binding of TNF, can be cleavedproteolytically and retrieved in the form of the soluble TNF-BP. Thepossibility should not be ruled out that, with regard to the operatingcapacity of the receptor, this protein may possibly be associated withone or more other proteins.

For the purposes of the production of TNF-BP on a larger scale, it isadvantageous not to start from the whole cDNA, since the need to cleaveTNF-BP from that part of the protein which represents the membrane-boundpart of the TNF receptor must be borne in mind. Rather, as mentionedhereinbefore, a translation stop codon is expediently inserted after thecodon for Asn-172 by controlled mutagenesis in order to prevent proteinsynthesis going beyond the C-terminal end of TNF-BP. With the cDNA whichis initially obtained according to the invention and which represents apartial sequence of the DNA coding for a TNF receptor, it is possible toobtain the complete receptor sequence by amplifying the missing 3′-end,e.g., by means of modified PCR (RACE=“rapid amplification of cDNA ends”;Frohman, M. A., et al., Proc. Natl. Acad. Sci. 85:8998–9002 (1988)),with the aid of a primer constructed on the basis of a sequence locatedas far as possible in the direction of the 3′-end of the cDNA present.An alternative method is the conventional screening of the cDNA librarywith the available cDNA or parts thereof as a probe.

According to the invention, first of all, the rat TNF receptor cDNA wasisolated and with a partial sequence therefrom the complete human TNFreceptor cDNA was obtained and brought to expression.

The invention relates to a human TNF receptor and the DNA coding for it.This definition also includes DNAs which code for C- and/or N-terminallyshortened, e.g., processed forms or for modified forms (e.g., by changesat proteolytic cleavage sites, glycosylation sites or specific domainregions) or for fragments, e.g., the various domains, of the TNFreceptor. These DNAs may be used in conjunction with the controlsequences needed for expression as a constituent of recombinant DNAmolecules, to which the present invention also relates, for transformingprokaryotic or eukaryotic host organisms. On the one hand this createsthe prerequisite for preparing the TNF receptor or modifications orfragments thereof in larger quantities by the recombinant method, inorder to make it possible for example to clarify the three-dimensionalstructure of the receptor. On the other hand, these DNAs can be used totransform higher eukaryotic cells in order to allow a study of themechanisms and dynamics of the TNF/receptor interaction, signaltransmission or the relevance of the various receptor domains orsections thereof.

The present invention encompasses the expression of the desired TNFbinding protein in either prokaryotic or eukaryotic cells. Preferredeukaryotic hosts include yeast (especially Saccharomyces), fungi(especially Aspergillus), mammalian cells (such as, for example, humanor primate cells) either in vivo or in tissue culture.

Yeast and mammalian cells are preferred hosts of the present invention.The use of such hosts provides substantial advantages in that they canalso carry out post-translational peptide modifications includingglycosylation. A number of recombinant DNA strategies exist whichutilize strong promoter sequences and high copy number of plasmids whichcan be utilized for production of the desired proteins in these hosts.

Yeast recognize leader sequences on cloned mammalian gene products andsecrete peptides bearing leader sequences (i.e., pre-peptides).Mammalian cells provide post-translational modifications to proteinmolecules including correct folding or glycosylation at correct sites.

Mammalian cells which may be useful as hosts include cells of fibroblastorigin such as VERO or CHO-Kl, and their derivatives. For a mammalianhost, several possible vector systems are available for the expressionof the desired TNF binding protein. A wide variety of transcriptionaland translational regulatory sequences may be employed, depending uponthe nature of the host. The transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, simian virus, or the like, where the regulatory signalsare associated with a particular gene which has a high level ofexpression. Alternatively, promoters from mammalian expression products,such as actin, collagen, myosin, etc., may be employed. Transcriptionalinitiation regulatory signals may be selected which allow for repressionor activation, so that expression of the genes can be modulated. Ofinterest are regulatory signals which are temperature-sensitive so thatby varying the temperature, expression can be repressed or initiated, orare subject to chemical regulation, e.g., metabolite.

The expression of the desired TNF binding protein in eukaryotic hostsrequires the use of eukaryotic regulatory regions. Such regions will, ingeneral, include a promoter region sufficient to direct the initiationof RNA synthesis. Preferred eukaryotic promoters include the promoter ofthe mouse metallothionein I gene (Hamer, D., et al., J. Mol. Appl. Gen.1:273–288 (1982)); the TK promoter of Herpes virus (McKnight, S., Cell31:355–365 (1982)); the SV40 early promoter (Benoist, C., et al., Nature(London) 290:304–310 (1981)); the yeast qal4 gene promoter (Johnston, S.A., et al., Proc. Natl. Acad. Sci. (USA) 79:6971–6975 (1982); Silver, P.A., et al., Proc. Natl. Acad. Sci. (USA) 81:5951–5955 (1984)).

As is widely known, translation of eukaryotic mRNA is initiated at thecodon which encodes the first methionine. For this reason, it ispreferable to ensure that the linkage between a eukaryotic promoter anda DNA sequence which encodes the desired receptor molecule does notcontain any intervening codons which are capable of encoding amethionine (i.e., AUG). The presence of such codons results either inthe formation of a fusion protein (if the AUG codon is in the samereading frame as the desired receptor molecule-encoding DNA sequence) ora frame-shift mutation (if the AUG codon is not in the same readingframe as the desired TNF binding protein-encoding sequence).

The expression of the TNF binding proteins can also be accomplished inprocaryotic cells. Preferred prokaryotic hosts include bacteria such asE. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc.The most preferred prokaryotic host is E. coli. Bacterial hosts ofparticular interest include E. coli K12 strain 294 (ATCC 31446), E. coliX1776 (ATCC 31537), E. coli W3110 (F⁻, lambda⁻, prototrophic; ATCC27325), and other enterobacteria (such as Salmonella typhimurium orSerratia marcescens), and various Pseudomonas species. The prokaryotichost must be compatible with the replicon and control sequences in theexpression plasmid.

To express the desired TNF binding protein in a prokaryotic cell (suchas, for example, E. coli, B. subtilis, Pseudomonas, Streptomyces, etc.),it is necessary to operably link the desired receptor molecule-encodingsequence to a functional prokaryotic promoter. Such promoters may beeither constitutive or, more preferably, regulatable (i.e., inducible orderepressible). Examples of constitutive promoters include the intpromoter of bacteriophage λ and the bla promoter of the β-lactamase geneof pBR322, etc. Examples of inducible prokaryotic promoters include themajor right and left promoters of bacteriophage λ (P_(L) and P_(R)), thetrp, recA, lacZ, lacI, qal, and tac promoters of E. coli, the α-amylase(Ulmanen, I., et al., J. Bacteriol. 162:176–182 (1985)), theσ-28-specific promoters of B. subtilis (Gilman, M. Z., et al., Gene32:11–20 (1984)), the promoters of the bacteriophages of Bacillus(Gryczan, T. J., In: The Molecular Biology of the Bacilli, AcademicPress, Inc., NY (1982)), and Streptomyces promoters (Ward, J. M., etal., Mol. Gen. Genet. 203:468–478 (1986)). Prokaryotic promoters arereviewed by Glick, B. R., (J. Ind. Microbiol. 1:277–282 (1987));Cenatiempo, Y. (Biochimie 68:505–516 (1986)); and Gottesman, S. (Ann.Rev. Genet. 18:415–442 (1984)).

Proper expression in a prokaryotic cell also requires the presence of aribosome binding site upstream from the gene-encoding sequence. Suchribosome binding sites are disclosed, for example, by Gold, L., et al.(Ann. Rev. Microbiol. 35:365–404 (1981)).

The desired TNF binding protein-encoding sequence and an operably linkedpromoter may be introduced into a recipient prokaryotic or eukaryoticcell either as a non-replicating DNA (or RNA) molecule, which may eitherbe a linear molecule or, more preferably, a closed covalent circularmolecule. Since such molecules are incapable of autonomous replication,the expression of the desired receptor molecule may occur through thetransient expression of the introduced sequence. Alternatively,permanent expression may occur through the integration of the introducedsequence into the host chromosome.

In one embodiment, a vector is employed which is capable of integratingthe desired gene sequences into the host cell chromosome. Cells whichhave stably integrated the introduced DNA into their chromosomes can beselected by also introducing one or more markers which allow forselection of host cells which contain the expression vector. The markermay complement an auxotrophy in the host (such as leu2, or ura3, whichare common yeast auxotrophic markers), biocide resistance, e.g.,antibiotics, or heavy metals, such as copper, or the like. Theselectable marker gene can either be directly linked to the DNA genesequences to be expressed, or introduced into the same cell byco-transfection.

In a preferred embodiment, the introduced sequence will be incorporatedinto a plasmid or viral vector capable of autonomous replication in therecipient host. Any of a wide variety of vectors may be employed forthis purpose. Factors of importance in selecting a particular plasmid orviral vector include: the ease with which recipient cells that containthe vector may be recognized and selected from those recipient cellswhich do not contain the vector; the number of copies of the vectorwhich are desired in a particular host; and whether it is desirable tobe able to “shuttle” the vector between host cells of different species.

Any of a series of yeast gene expression systems can be utilized.Examples of such expression vectors include the yeast 2-micron circle,the expression plasmids YEP13, YCP, and YRP, etc., or their derivatives.Such plasmids are well known in the art (Botstein, D., et al., MiamiWntr. Symp. 19:265–274 (1982); Broach, J. R., In: The Molecular Biologyof the Yeast Saccharomyces: Life Cycle and Inheritance, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., pp. 445–470 (1981); Broach,J. R., Cell 28:203–204 (1982)).

For a mammalian host, several possible vector systems are available forexpression. One class of vectors utilize DNA elements which provideautonomously replicating extra-chromosomal plasmids, derived from animalviruses such as bovine papilloma virus, polyoma virus, adenovirus, orSV40 virus. A second class of vectors relies upon the integration of thedesired gene sequences into the host chromosome. Cells which have stablyintegrated the introduced DNA into their chromosomes may be selected byalso introducing one or more markers which allow selection of host cellswhich contain the expression vector. The marker may provide forprototropy to an auxotrophic host, biocide resistance, e.g.,antibiotics, or heavy metals, such as copper, or the like. Theselectable marker gene can either be directly linked to the DNAsequences to be expressed, or introduced into the same cell byco-transformation. Additional elements may also be needed for optimalsynthesis of mRNA. These elements may include splice signals, as well astranscription promoters, enhancers, and termination signals. The cDNAexpression vectors incorporating such elements include those describedby Okayama, H., Mol. Cell. Biol. 3:280 (1983), and others.

Preferred prokaryotic vectors include plasmids such as those capable ofreplication in E. coli, such as, for example, pBR322, ColEl, pSC101,pACYC 184, and πVX. Such plasmids are, for example, disclosed byManiatis, T., et al. (In: Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1982)). Bacillus plasmidsinclude pC194, pC221, and pT127, etc. Such plasmids are disclosed byGryczan, T. (In: The Molecular Biology of the Bacilli, Academic Press,NY (1982), pp. 307–329). Suitable Streptomyces plasmids include pIJ101(Kendall, K. J., et al., J. Bacteriol. 169:4177–4183 (1987)), andStreptomyces bacteriophages such as φC31 (Chater, K. F., et al., In:Sixth International Symposium on Actinomycetales Biology, AkademiaiKaido, Budapest, Hungary (1986), pp. 45–54). Pseudomonas plasmids arereviewed by John, J. F., et al. (Rev. Infect. Dis. 8:693–704 (1986)),and Izaki, K. (Jpn. J. Bacteriol. 33:729–742 (1978)).

Once the vector or DNA sequence containing the constructs has beenprepared for expression, the DNA constructs may be introduced into anappropriate host. Various techniques may be employed, such as protoplastfusion, calcium phosphate precipitation, electroporation, or otherconventional techniques. After the fusion, the cells are grown in mediaand screened for appropriate activities. Expression of the sequenceresults in the production of the TNF binding protein.

The TNF binding proteins of the invention may be isolated and purifiedfrom the above-described recombinant molecules in accordance withconventional methods, such as extraction, precipitation, chromatography,affinity chromatography, electrophoresis, or the like. The term“substantially pure” is intended to mean TNF binding proteins which aresubstantially one major band by SDS-polyacrylamide gel electrophoresisand which contain only minor amounts of other proteins which wouldnormally contaminate a whole cell lysate containing native TNF receptorprotein, as evidenced by the presence of other minor bands.

The recombinant TNF receptor (or fragments or functional derivativesthereof) can be used to investigate substances for their interactionwith TNF or the TNF receptor or their influence on the signaltransmission induced by TNF. Such screenings (usingproteins/fragments/variants or suitably transformed higher eukaryoticcells) create the prerequisite for the identification of substanceswhich substitute TNF, inhibit the binding thereof to the receptor orthose which are capable of blocking or intensifying the mechanism ofsignal transmission initiated by TNF.

One possible method of discovering agonists and antagonists of TNF orthe TNF receptor is in the establishment of high capacity screening. Asuitable cell line, preferably one which does not express endogenoushuman TNF receptor, is transformed with a vector which contains the DNAcoding for a functional TNF receptor and optionally modified from thenatural sequence. The activity of agonists or antagonists can beinvestigated in screening of this kind by monitoring the response to theinteraction of the substance with the receptor using a suitable reporter(altered enzyme activity, e.g., protein kinase C, or gene activation,e.g., manganese superoxide dismutase, NF-KB). Investigations into themechanisms and dynamics of the TNF/receptor interaction, signaltransmission, or the role of the receptor domains in this respect mayalso be carried out, for example, by combining DNA fractions coding forthe extracellular domain of the TNF receptor (or parts thereof) with DNAfractions coding for various transmembrane domains and/or variouscytoplasmatic domains and bringing them to expression in eukaryoticcells. The hybrid expression products which may be obtained in this waymay be capable of giving conclusive information as to the relevance ofthe various receptor domains, on the basis of any changes in theproperties for signal transduction, so that targeted screening is madeeasier.

The availability of the cDNA coding for the TNF receptor or fractionsthereof is the prerequisite for obtaining the genomic DNA. Understringent conditions, a DNA library is screened and the clones obtainedare investigated to see whether they contain the regulatory sequenceelements needed for gene expression in addition to the coding regions(e.g., checking for promoter function by fusion with coding regions ofsuitable reporter genes). Methods for screening DNA libraries understringent conditions are taught, for example, in EP-A 0 174 143,incorporated by reference herein. Obtaining the genomic DNA sequencemakes it possible to investigate the regulatory sequences situated inthe area which does not code for the TNF receptor, particularly in the5-flanking region, for any possible interaction with known substanceswhich modulate gene expression, e.g., transcription factors or steroids,or possibly discover new substances which might have a specific effecton the expression of this gene. The results of such investigationsprovide the basis for the targeted use of such substances for modulatingTNF receptor expression and hence for directly influencing the abilityof the cells to interact with TNF. As a result, the specific reactionwith the ligands and the resulting effects can be suppressed.

The scope of the present invention also includes DNAs which code forsubtypes of the TNF receptor or its soluble forms, which may possiblyhave properties different from those of the present TNF receptor. Theseare expression products which are formed by alternative splicing andhave modified structures in certain areas, e.g., structures which canbring about a change in the affinity and specificity for the ligand(TNF-α/TNF-β) or a change in terms of the nature and efficiency ofsignal transmission.

With the aid of the cDNA coding for the TNF receptor it is possible toobtain nucleic acids which hybridize with the cDNA or fragments thereofunder conditions of low stringency and code for a polypeptide capable ofbinding TNF or contain the sequence coding for such a polypeptide.

According to a further aspect the invention relates to recombinantTNF-BP, preferably in a secretable form, which constitutes the solublepart of the TNF receptor according to the invention, and the DNA codingfor it. By introducing a DNA construct containing the sequence codingfor TNF-BP with a sequence coding for a signal peptide under the controlof a suitable promoter into suitable host organisms, especiallyeukaryotic and preferably higher eukaryotic cells, it is possible toproduce TNF-BP which is secreted into the cell supernatant.

If a signal peptide is used with regard to the secretion of the protein,the DNA coding for the signal peptide is conveniently inserted beforethe codon for Asp-12 in order to obtain a uniform product.Theoretically, any signal peptide is suitable which guarantees secretionof the mature protein in the corresponding host organism. If necessary,the signal sequence can also be placed in front of the triplet codingfor Leu-1; in this case, it may be necessary to separate the form ofTNF-BP produced by splitting off the peptide which consists of 11 aminoacids at the N-terminus, from the unprocessed or incompletely processedTNF-BP in an additional purification step.

Since the cDNA after the codon for Asn-172, which represents theC-terminus on the basis of C-terminal analysis, does not contain a stopcodon, a translation stop codon is expediently introduced, with respectto the expression of TNF-BP, after the codon for Asn-172, by controlledmutagenesis.

The DNA coding for TNF-BP can be modified by mutation, transposition,deletion, addition, or truncation provided that DNAs modified in thisway code for (poly)peptides capable of binding TNF. Such modificationsmay consist, for example, of changing one or more of the potentialglycosylation sites which are not necessary for the biological activity,e.g., by replacing the Asn codon by a triplet which codes for adifferent amino acid. With a view to maintaining the biologicalactivity, modifications which result in a change in the disulfidebridges (e.g., a reduction in their number) may also be carried out.

The DNA molecules referred to thus constitute the prerequisite forconstructing recombinant DNA molecules, which are also an object of theinvention. With recombinant DNA molecules of this kind in the form ofexpression vectors containing the DNA, optionally suitably modified,which codes for a protein with TNF-BP activity, preferably with apreceding signal sequence, and the control sequences needed forexpression of the protein, it is possible to transform and cultivatesuitable host organisms and obtain the protein.

Just like any modifications to the DNA sequence, host cells or organismssuitable for expression are selected particularly with regard to thebiological activity of the protein in binding TNF. Furthermore, thecriteria which are conventionally applied to the preparation ofrecombinant proteins such as compatibility with the chosen vector,processability, isolation of the protein, expression characteristics,safety, and cost aspects are involved in the decision as to the hostorganism. The choice of a suitable vector arises from the host intendedfor transformation. In principle, all vectors which replicate andexpress the DNAs (or modifications thereof) coding for TNF-BP accordingto the invention are suitable.

With respect to the biological activity of the protein, in theexpression of the DNA coding for TNF-BP, particular account should betaken of any relevance of the criteria, found in the natural protein, ofglycosylation and a high proportion of cysteine groups to the propertyof binding TNF. Conveniently, therefore, eukaryotes, particularlysuitable expression systems of higher eukaryotes, are used for theexpression.

Within the scope of the present invention, both transient and permanentexpression of TNF-BP were demonstrated in eukaryatic cells.

The recombinant TNF-BP according to the invention and suitablemodifications thereof which have the capacity to bind TNF can be used inthe prophylactic and therapeutic treatment of humans and animals forindications in which a harmful effect of TNF-α occurs. Since TNF-BP hasalso been shown to have a TNF-β inhibiting activity, it (or theassociated or modified polypeptides) can be used in suitable doses,possibly in a form modified to give a greater affinity for TNF-β, toinhibit the effect of TNF-β in the body.

The invention therefore also relates to pharmaceutical preparationscontaining a quantity of recombinant TNF-BP which effectively inhibitsthe biological activity of TNF-α and/or TNF-β or a related polypeptidecapable of binding TNF.

Pharmaceutical preparations are particularly suitable for parenteraladministration for those indications in which TNF displays a harmfuleffect, e.g., in the form of lyophilized preparations or solutions.These contain TNF-BP or a therapeutically active functional derivativethereof in a therapeutically active amount, optionally together withphysiologically acceptable additives such as stabilizers, buffers, andpreservatives, etc.

The dosage depends particularly on the indication and the specific formof administration, e.g., whether it is administered locally orsystemically. The size of the individual doses will be determined on thebasis of an individual assessment of the particular illness, taking intoaccount such factors as the patient's general health, anamnesis, age,weight, and sex, etc. It is essential when determining thetherapeutically effective dose to take into account the quantity of TNFsecreted which is responsible for the disease as well as the quantity ofendogenous TNF-BP. Basically, it can be assumed that, for effectivetreatment of a disease triggered by TNF, at least the same molar amountof TNF-BP is required as the quantity of TNF secreted, and possibly amultiple excess might be needed.

More specifically, the objective of the invention is achieved asfollows:

The N-terminal amino acid sequence of the highly purified TNF-BP and theamino acid sequences of peptides obtained by tryptic digestion of theprotein can be determined.

Moreover, the C-terminus was determined by carboxy-peptidase Pdigestion, derivatization of the amino acids split off andchromatographic separation. From the peptide sequences obtained bytryptic digestion, with a view to their use in PCR for the preparationof oligonucleotides, regions were selected from the N-terminus on theone hand and from a tryptic peptide on the other hand such that thecomplexity of mixed oligonucleotides for hybridization with cDNA is keptto a minimum. A set of mixed oligonucleotides were prepared on the basisof these two regions, the set derived from the region located at theN-terminus being synthesized in accordance with mRNA, whilst the setderived from the tryptic peptide was synthesized in reverse, so as to becomplementary to the mRNA. In order to facilitate the subsequent cloningof a segment amplified with PCR, the set of oligonucleotides derivedfrom the tryptic peptide was given a BamHI restriction site. Then λ DNAwas isolated from the TNF-α induced fibrosarcoma cDNA library and fromthis a TNF-BP sequence was amplified using PCR. The resulting fragmentwas cloned and sequenced; it comprises 158 nucleotides and contains thesequence coding for the tryptic peptide 20 between the two fragments ofsequence originating from the primer oligonucleotides.

This DNA fragment was subsequently radioactively labeled and used as aprobe for isolating cDNA clones from the fibrosarcoma library. Theprocedure involved first hybridizing plaques with the probe, separatingphages from hybridizing plaques and obtaining λ DNA therefrom.Individual cDNA clones were subcloned and sequenced; two of thecharacterized clones contained the sequence coding for TNF-BP.

This sequence constitutes part of the sequence coding for a TNFreceptor.

After shortening of the 5′-non-coding region and insertion of a stopcodon after the codon for the C-terminal amino acid of the naturalTNF-BP, the cDNA was inserted in a suitable expression plasmid,eukaryotic cells were transformed therewith, and the expression ofTNF-BP was demonstrated using ELISA.

The still outstanding 3′-region of the TNF receptor was obtained bysearching through a rat brain cDNA library from the rat glia tumour cellline C6 using a TNF-BP probe and isolating all the cDNA coding for therat TNF receptor.

The fraction of this cDNA at the 3′-end, which was assumed to correspondto the missing 3′-region behind the EcoRI cutting site of the human TNFreceptor, was used as a probe to search through the HS913T cDNA libraryonce more. A clone was obtained which contains all the DNA coding forthe TNF receptor.

After shortening of the 5′-non-coding region, the cDNA was inserted inan expression plasmid and the expression of human TNF receptor wasdemonstrated in eukaryotic cells by means of the binding ofradioactively labeled TNF.

Northern blot analysis confirmed that the isolated cDNA correspondssubstantially to all the TNF-R mRNA (the slight discrepancy arises fromthe absence of part of the 5′-non-coding region). From this it can beconcluded that the expressed protein is the complete TNF receptor.

The invention is illustrated by means of the Examples which follow.

EXAMPLE 1

Preparation of Highly Purified TNF-BP

a) Concentration of Urine

200 liters of dialyzed urine from uraemia patients, stored in flaskscontaining EDTA (10 g/l), Tris (6 g/l), NaN₃ (1 g/l), and benzamidinehydrochloride (1 g/l) and kept in a refrigerator were concentrated byultrafiltration using a highly permeable haemocapillary filter with anasymmetric hollow fibre membrane (FH 88H, Gambro) down to 4.2 literswith a protein content of 567 g. The concentrated urine was dialyzedagainst 10 mM/l Tris-HCl, pH 8. During this procedure, as in thefollowing steps (except reverse phase chromatography), 1 mM/l ofbenzamidine hydrochloride was added in order to counteract proteolyticdigestion. Unless otherwise stated, all the subsequent purificationsteps were carried out at 4° C.

b) Ion Exchange Chromatography

This step was carried out by charging DEAE Sephacel columns (2.5×40 cm)with samples of concentrated and dialyzed urine containing about 75 g ofprotein. Elution was carried out with 800 ml of a NaCl/10 mM Tris-HCl,pH 8 gradient, the NaCl concentration being 0 to 0.4 M. The fractionsfrom seven columns containing the TNF-BP with a total protein content of114 g were stored at −20° C.

c) Affinity Chromatography

In order to prepare the TNF Sepharose column, rTNF-α (15 mg) in 0.1 MNaHCO₃, 1 M NaCl, pH 9 (coupling buffer) was coupled to 1.5 g ofcyanogen bromide-activated Sepharose 4B (Pharmacia). The Sepharose wasswelled in 1 mM HCl and washed with coupling buffer. After the additionof rTNF-α the suspension was left to rotate for 2 hours at ambienttemperature. The excess CNBr groups were blocked by rotation for one anda half hours with 1M ethanolamine, pH 8. The TNF Sepharose was washed afew times alternately in 1M NaCl, 0.1 M sodium acetate, pH 8 and 1 MNaCl, 0.1 M boric acid, pH 4 and then stored in phosphate-bufferedsaline solution with 1 mM benzamidine hydrochloride. The fractionsobtained from step b) were adjusted to a concentration of 0.2 M NaCl, 10mM Tris-HCl, pH 8. The TNF-Sepharose was packed into a column and washedwith 0.2 M NaCl, 10 mM Tris-HCl, pH 8 and the TNF-BP-containingfractions, corresponding to about 30 g of protein, were applied at athroughflow rate of 10 ml/h and washed exhaustively with 0.2 M NaCl, 10mM Tris-HCl, pH 8, until no further absorption could be detected in theeluate at 280 nm. Then TNF-BP was eluted with 0.2 M glycine/HCl, pH 2.5.TNF-BP-containing fractions from 4 separations were combined andlyophilized after the addition of polyethylene glycol (MW 6000) up to afinal concentration of 10 mg/ml. The lyophilized sample was dissolved indistilled water and dialyzed against distilled water (The dialyzedsample (4 ml) was stored in deep-frozen state).

This purification step further concentrated the product by about 9000times compared with the previous product. SDS-PAGE (carried out asdescribed in preliminary test 2) of the TNF-BP containing fractionsshowed the elution of three main components with molecular weights of28,000; 30,000; and 50,000.

d) Reverse Phase Chromatography

An aliquot (1 ml) of the fractions obtained from step c) with theaddition of 0.1% trifluoroacetic acid was applied to a ProRPC HR 5/10column (Pharmacia), connected to an FPLC system (Pharmacia). The columnwas equilibrated with 0.1% trifluoroacetic acid and charged at ambienttemperature with a linear 15 ml gradient of 10 vol % to 50 vol %aceto-nitrile containing 0.1% trifluoroacetic acid; the through-flowrate was 0.3 ml/min. Fractions of 0.5 ml were collected and theabsorption at 280 nm was determined, as well as the activity of theTNF-α binding protein, using the competitive binding test as describedin Example 5, using 0.01 μl of sample in each case. TNF-BP eluted as asingle activity peak corresponding to a sharp UV absorption peak.

This last purification step brought an increase in specific activity ofabout 29 fold, whilst the total increase in activity compared with thestarting material (concentrated dialysis urine) was about 1.1×10⁶-fold.SDS-PAGE of the reduced and non-reduced samples, carried out asdescribed in preliminary test 2, resulted in a diffuse band, indicatingthe presence of a single polypeptide with a molecular weight of about30,000. The diffused appearance of the band may be due to the presenceof one or more heterogeneous glycosylations and/or a second polypeptidepresent in a smaller amount. The assumption that it might be apolypeptide with the N-terminus found to be a secondary sequence inpreliminary test 3d), which is longer than TNF-BP at the N-terminus, wasconfirmed by the sequence of the cDNA, according to which there is afraction of 11 amino acids between the signal sequence and Asn (position12), the sequence of which coincides with the N-terminal secondarysequence and which is obviously split off from the processed protein.

EXAMPLE 2

SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE was carried out using the method of Laemmli (Laemmli, U. K.,Nature 227:680–684 (1970)) on flat gels measuring 18 cm long, 16 cmwide, and 1.5 mm thick, with 10 pockets, by means of an LKB 2001electrophoresis unit. The protein content of the samples from thepurification steps c) and d) (preliminary test 1) was determined byBio-Rad Protein Assay or calculated from the absorption at 280 nm, anabsorption of 1.0 being recognized to be equivalent to a content of 1 mgTNF-BP/ml.

The samples containing about 25 μg of protein (from preliminary test 1c)or about 5 μg (from 1 d) in reduced form (β-mercaptoethanol) andnon-reduced form, were applied to a 3% collecting gel and a 5 to 20%linear polyacrylamide gradient gel. Electrophoresis was carried out at25 mA/gel without cooling. The molecular weight markers used (Pharmacia)were phosphorylase B (MW 94,000), bovine serum albumin (MW 67,000),ovalbumin (MW 43,000), carboanhydrase (MW 30,000), soya bean trypsininhibitor (MW 20,100), and α-lactalbumin (MW 14,400). The gels werestained with Coomassie Blue in 7% acetic acid/40% ethanol anddecolorized in 7% acetic acid/25% ethanol.

The results of the SDS-PAGE showed TNF-BP to be a polypeptide chain witha molecular weight of about 30,000.

EXAMPLE 3

a) Preparation of Samples

15 μg of the protein purified according to preliminary test 1d) weredesalinated using reverse phase HPLC and further purified. To do this aBakerbond WP C18 column was used (Baker; 4.6×250 mm) and 0.1%trifluoroacetic acid in water (eluant A) or in acetonitrile (eluant B)as the mobile phase. The increase in the gradient was 20 to 68% eluant Bin 24 minutes. Detection was carried out in parallel at 214 nm and 280nm. The fraction containing TNF-BP was collected, dried, and dissolvedin 75 μl of 70% formic acid and used directly for the amino acidsequence analysis.

b) Amino Acid Sequence Analysis

The automatic amino acid sequence analysis was carried out with anApplied Biosystems 477 A liquid phase sequenator by on-linedetermination of the phenylthiohydantoin derivatives released, using anApplied Biosystems Analyser, Model 120 A PTH. It gave the followingN-terminal sequence as the main sequence (about 80% of the quantity ofprotein): Asp-Ser-Val-Xaa-Pro-Gln-Gly-Lys-Tyr-Ile-His-Pro-Gln (SEQ IDNO: 28). In addition, the following secondary sequence was detected:Leu-(Val)-(Pro)-(His)-Leu-Gly-Xaa-Arg-Glu (SEQ ID NO: 29). The aminoacids shown in brackets could not be clearly identified.

EXAMPLE 4

SDS-PAGE

The sample was prepared as described in Example 3 with the differencethat the quantity of sample was 10 μg. The sample was taken up in 50 μlof water and divided into 4 portions. One of the four aliquot parts wasreduced in order to determine its purity by SDS-PAGE according to themethod of Laemmli (Laemmli, U. K., Nature 227:680–684 (1970)) with DTT(dithiothreitol) and separated on minigels (Hofer, 55×80×0.75 mm, 15%);the molecular weight marker used was the one specified in Example 2.Staining was carried out using the Oakley method (Oakley, B. R., et al.,Analyt. Biochem. 105:361–363 (1986)). The electropherogram showed asingle band having a molecular weight of about 30,000.

EXAMPLE 5

a) Tryptic Peptide Mapping

About 60 μg of the protein purified in Example 1d) was desalinated byreverse phase HPLC and further purified thereby. A Bakerbond WP C18column (Baker; 4.6×250 mm) was used, and 0.1% trifluoroacetic acid inwater (eluant A) or in acetonitrile (eluant B) was used as the mobilephase. The increase in gradient amounted to 20 to 68% eluant B in 24minutes. Detection was carried out in parallel at 214 nm and at 280 nm.The fraction containing TNF-BP (retention time about 13.0 min.) wascollected, dried, and dissolved in 60 μl of 1% ammonium bicarbonate.

1% w/w, corresponding to 0.6 μg of trypsin (Boehringer Mannheim) wasadded to this solution and the reaction mixture was incubated for 6hours at 37° C. Then a further 1% w/w of trypsin was added andincubation was continued overnight.

In order to reduce the disulfide bridges, the reaction mixture was thencombined with 60 μl of 6 M urea and with 12 μl of 0.5 M dithiothreitoland left to stand for 2 hours at ambient temperature.

The tryptic cleavage peptides produced were separated by reverse phaseHPLC, using a Delta Pak C18 column (Waters, 3.9×150 mm, 5 μm particlediameter, 100 A pore diameter) at 30° C. and 0.1% trifluoroacetic acidin water (eluant A) or in acetonitrile (eluant B) as the mobile phase.The gradient was increased from 0 to 55% of eluant B in 55 minutes, then55% B was maintained for 15 minutes. The flow rate was 1 ml/min. anddetection was carried out in parallel at 214 nm (0.5 AUFS) and at 280 nm(0.05 AUFS).

b) Sequence Analysis of Tryptic Peptides

Some of the tryptic cleavage peptides of TNF-BP obtained in a) weresubjected to automatic amino acid sequence analysis. The correspondingfractions from reverse phase HPLC were collected, dried, and dissolvedin 75 μl of 70% formic acid. These solutions were used directly forsequencing in an Applied Biosystems 477 A Pulsed Liquid PhaseSequenator. Table 2 contains the results of the sequence analysis of thetryptic peptides (the amino acids shown in brackets could not beidentified with certainty). The letters “Xaa” indicate that at thispoint the amino acid could not be identified. In fraction 8 the aminoacid in position 6 could not be identified. The sequence Xaa-Asn-Ser forposition 6–8 leads one to suppose that the amino acid 6 is present inglycosylated form.

In fraction 17 the amino acid in position 6 could not be identifiedeither. The sequence Xaa-Asn-Ser (already occurring in fraction 8) forpositions 6–8 leads one to suppose that amino acid 6 is present inglycosylated form. The first 13 amino acids of fraction 17 aresubstantially identical to fraction 8; fraction 17 should thus be apeptide formed by incomplete tryptic cleavage.

It is striking that fraction 21 is identical to positions 7 to 14 offraction 27. Both in fraction 21 and in fraction 27 the sequencesuddenly breaks off after the amino acid asparagine (position 8 or 14),even though no tryptic cleavage can be expected here. This indicatesthat the amino acid asparagine position 8 in fraction 21 or position 14in fraction 27) could be the C-terminal amino acid of TNF-BP.

It is noticeable that the sequence of fraction 12 which occurs only insmall amounts, is substantially identical to the secondary sequence ofthe N-terminus found in preliminary test 10. The fact that the proteinsof the main and subsidiary sequence could not be separated on ananalytical reverse phase HPLC column (Example 3b) indicated that theprotein with the subsidiary sequence was a form of TNF-BP extended atthe N-terminus, which was largely converted by processing into theprotein with the main sequence.

TABLE 1 Amino acid sequences of the analyzed tryptic peptides of TNF-BPFraction Amino acid sequence 1 Asp - Ser - Val - Cys - Pro - Gln - Gly -Lys 2 Xaa - Xaa - Leu - Ser -(Cys)- Ser - Lys 3 Asp - Thr - Val -(Cys)-Gly -(Cys)- Arg 4 Glu - Asn - Glu -(Cys)- Val - Ser -(Cys)- Ser - Asn-(Cys)- Lys 5 Glu - Asn - Glu -(Cys)- Val - Ser - (Cys)-(Ser)- Asn-(Cys)- Lys -(Lys) 8 Tyr - Ile - His - Pro - Gln - Xaa - Asn - Ser -Ile - Xaa - Xaa - Xaa - Lys 11 Glu - Cys -Glu - Ser - Gly - Ser - Phe -Thr - Ala - Ser - Glu - Asn -(Asn)-(Lys) 12 Leu - Val - Pro - His -Leu - Gly - Asp - Arg 13 Lys - Glu - Met - Gly - Gln - Val - Glu - Ile -Ser - Ser -(Cys)- Thr - Val - Asp - (Arg) 14/I  Gly - Thr - Tyr - Leu -Tyr - Asn - Asp - Cys - Pro - Gly - Pro - Gly - Gln - 14/II (Glu)- Met -Gly - Gln - Val -(Glu)-(Ile)- (Ser)- Xaa - Xaa - Xaa -(Val)-(Asp)- 15Lys - Glu - Met - Gly - Gln - Val - Glu - lie - Ser - Ser -(Cys)- Thr -Val - Asp - Arg Asp - Thr - Val -(Cys)- Gly - 17 Tyr - Ile - His - Pro -Gln - Xaa - Asn - Ser - Ile -(Cys)-(Cys)- Thr - Lys -(Cys) His - Lys -Gly - Xaa - Tyr - 20 Gly - Thr - Tyr - Leu - Tyr - Asn - Asp - Cys -Pro - Gly - Pro - Gly - Gln - Asp - Thr -Xaa - Xaa - Arg 21 Leu -(Cys)-Leu - Pro - Gln - Ile - Glu - Asn 26 Gln - Asn - Thr - Val -(Cys)- Thr -Xaa - (His)- Ala - Gly - Phe -(Phe)- Leu -(Arg) 27 Ser - Leu - Glu-(Cys)- Thr - Lys - Leu - (Cys)- Leu - Pro - Gln - Ile - Glu - Asn

EXAMPLE 6

Analysis of the C-Terminus

This analysis was carried out on the principle of the method describedin Hsieng, S. L., et al., J. Chromatography 447:351–364 (1988).

About 60 μg of the protein purified in Example 2d was desalinated andthus further purified by reverse phase HPLC. A Bakerbond WP C18 column(Baker; 4.6×250 mm) was used and 0.1% trifluoroacetic acid in water(eluant A) or in acetonitrile (eluant B) was used as the mobile phase.The gradient was increased from 20 to 68% eluant 8 in 24 minutes.Detection was carried out in parallel at 214 nm and at 280 nm. Thefraction containing TNF-BP (retention time about 13.0 min.) wascollected, dried, and dissolved in 120 μl of 10 mM sodium acetate(adjusted to pH 4 with 1 N HCl).

To this solution were added 6 μl of Brij 35 (10 mg/ml in water) and 1.5μl of carboxypeptidase P (0.1 mg/ml in water; Boehringer Mannheim, No.810142). This corresponds to a weight ratio of enzyme to protein of 1 to400 (Frohman, M. A., et al., Proc. Natl. Acad. Sci. 85:8998–9002(1988)).

Immediately after the addition of the enzyme a sample of 20 μl of thereaction mixture was taken and the enzymatic reaction therein wasstopped by acidifying with 2 μl of concentrated trifluoroacetic acid andby freezing at −20° C.

The reaction mixture was left to stand in a refrigerator (about 8° C.)and samples of 20 μl were taken after 10, 20, 60, and 120 minutes. Theremainder of the reaction mixture was left at ambient temperature foranother 120 minutes. Immediately after being taken, all the samples wereacidified by the addition of 2 μl of concentrated trifluoroacetic acidand frozen at −20° C., thereby interrupting the enzymatic reaction.

Parallel to the sample mixture described, containing about 60 μg ofTNF-BP, a reagent double blind control was set up under identicalconditions but with no protein added.

After the last sample had been taken all the samples were dried for 30minutes in a Speed Vac Concentrator, mixed with 10 μl of a solution of 2parts of ethanol, 2 parts of water, and 1 part of triethylamine(=“Redrying solution” of the Picotag amino acid analysis system ofWaters) and briefly dried again. Then the samples were each mixed with20 μl of the derivatization reagent(7:1:1:1=ethanol:water:triethylamine:phenylisothiocyanate; Picotagsystem) in order to derivatize the amino acids split off from theC-terminus, then left to stand for 20 minutes at ambient temperature,and then dried for 1 hour in a Speed Vac Concentrator.

In order to analyze the derivatized amino acids, the samples weredissolved in 100 μl of “Sample Diluent” (Picotag system of Waters). Ofthese solutions, 50 μl was analyzed by reverse phase HPLC (column,mobile phase, and gradient according to the original specifications ofthe Picotag system of Waters). The chromatograms of the samples andreagent double blind controls were compared with the chromatogram of asimilarly derivatized mixture (100 pmol/amino acid) of standard aminoacids (Beckman).

As can be seen from the quantitative results of the Picotag amino acidanalysis (Table 3), asparagine is very likely the C-terminal amino acidof TNF-BP. Apart from asparagine, glutamic acid and a smaller amount ofisoleucine were also detected after 240 minutes reaction. Quantities ofother amino acids significantly above the reagent double blind valuecould not be found even after 240 minutes reaction. This result(Ile-Glu-Asn as the C-terminus) confirms the supposition made from theN-terminal sequencing of the tryptic peptides 21 and 27, to the effectthat the amino acids identified at the C-terminus in these peptides(Ile-Glu-Asn; Example 5b) constitute the C-terminus of TNF-BP.

TABLE 2 Quantitative evaluation of the Picotag amino acid analysis afterreaction of carboxypeptidase P with TNF-BP Integrator units for theamino acids Reaction time Isoleucine Glutamic acid Asparagine 0 — — — 10— — — 20 — — 83.304 60 — — 168.250 120 — — 319.470 240 85.537 52.350416.570Methods Used in Examples 7 to 20:

In the Examples which follow, standard molecular biological methods wereused unless expressly stated otherwise, which can be found in therelevant textbooks or which correspond to the conditions recommended bythe manufacturers. To simplify the description of the Examples whichfollow, frequently recurring methods or designations are abbreviated:

“Cutting” or “digestion” of DNA refers to the catalytic cleaving of theDNA using restriction endonucleases (restriction enzymes) at sitesspecific to them (restriction sites). Restriction endonucleases arecommercially available and are used under the conditions recommended bythe manufacturers (buffer, bovine serum albumin (BSA) as carrierprotein, dithiothreitol (DTT) as antioxidant). Restriction endonucleasesare designated by a capital letter, usually followed by small lettersand normally a Roman numeral. The letters depend on the microorganismfrom which the restriction endonuclease in question was isolated (e.g.,Sma I: Serratia marcescens). Usually, about 1 μg of DNA is cut with oneor more units of the enzyme in about 20 μl of buffer solution. Normally,an incubation period of 1 hour at 37° C. is used, but this can be variedin accordance with the manufacturer's instructions for use. Aftercutting, the 5′-phosphate group is sometimes removed by incubation withalkaline phosphatase from calf intestine (CIP). This serves to preventan undesirable reaction of the specific site in a subsequent ligasereaction (e.g., circularization of a linearized plasmid without theinsertion of a second DNA fragment). Unless otherwise stated, DNAfragments are normally not dephosphorylated after cutting withrestriction endonucleases. Reaction conditions for incubation withalkaline phosphatase can be found, for example, in the M13 Cloning andSequencing Handbook (Amersham, PI/129/83/12). After the incubation,protein is removed by extraction with phenol and chloroform, and the DNAis precipitated from the aqueous phase by the addition of ethanol.

“Isolation” of a specific DNA fragment means the separation of the DNAfragments obtained by restriction digestion, e.g., on a 1% agarose gel.After electrophoresis and rendering the DNA visible in UV light bystaining with ethidium bromide (EtBr), the desired fragment is locatedby means of molecular weight markers which had been applied, and isbound by further electrophoresis on DE 81 paper (Schleicher and Schüll).The DNA is washed by rinsing with low salt buffer (200 mM NaCl, 20 mMTris, pH 7.5, 1 mM EDTA) and then is eluted with a high salt buffer (1 MNaCl, 20 mM Tris, pH 7.5, 1 mM EDTA). The DNA is precipitated by theaddition of ethanol.

“Transformation” means the introduction of DNA into an organism so thatthe DNA can be replicated therein, either extrachromosomally orchromosomally integrated. Transformation of E. coli follows the methodspecified in the M13 Cloning and Sequencing Handbook (Amersham,PI/129/83/12).

“Sequencing” of a DNA means the determination of the nucleotidesequence. To do this, first of all, the DNA which is to be sequenced iscut with various restriction enzymes and the fragments are introducedinto suitably cut M13 mp8, mp9, mp18, or mp19 double-stranded DNA, orthe DNA is fragmented by ultrasound, the ends repaired, and thesize-selected fragments introduced into Sma I cut, dephosphorylated M13mp8 DNA (Shotgun method). After transformation of E. coli JM 101,single-stranded DNA is isolated from recombinant M13 phages inaccordance with the M13 Cloning and Sequencing Handbook (Amersham,PI/129/83/12) and sequenced by the dideoxy method (Sanger et al., Proc.Natl. Acad. Sci. 74:5463–5467 (1977)). As an alternative to the use ofthe Klenow fragment of E. coli DNA polymerase I, it is possible to useT7-DNA polymerase (“Sequenase”; United States Biochemical Corporation).The sequence reactions are carried out in accordance with the manual“Sequenase: Step-by-Step Protocols for DNA Sequencing With Sequenase”(Version 2.0).

Another method of sequencing consists of cloning the DNA which is to besequenced into a vector which carries, inter alia, a replication originof a DNA single-strand phage (M13, f1) (e.g., Bluescribe or BluescriptM13; Stratagene). After transformation of E. coli JM101 with therecombinant molecule, the transformants can be infected with a helperphage, e.g., M13KO7 or R408; Promega). As a result, a mixture of helperphages and packaged, single-stranded recombinant vector is obtained. Thesequencing template is worked up analogously to the M13 method.Double-stranded plasmid DNA is denatured by alkali treatment anddirectly sequenced in accordance with the above-mentioned sequencinghandbook.

The sequences were evaluated using the computer programs originallydeveloped by Staden (Staden, R., Nucleic Acid Res. 10:4731–4751 (1982))and modified by Pieler (Pieler, C., Dissertation, Universität Wien(1987)). “Ligating” refers to the process of forming phosphodiesterbonds between two ends of double-strand DNA fragments. Usually, between0.02 and 0.2 μg of DNA fragments in 10 μl are ligated with about 5 unitsof T4 DNA ligase (“ligase”) in a suitable buffer solution (Maniatis, T.,et al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, p. 474 (1982)). “Excising” of DNA from transformants refersto the isolation of the plasmid DNA from bacteria by the alkaline SDSmethod, modified according to Birnboim and Doly, leaving out thelysozyme. From 1.5 to 50 ml of bacterial culture is used.

“Oligonucleotides” are short polydeoxynucleotides which are chemicallysynthesized. The Applied Biosystems Systems Synthesizer Model 381A isused for this. The oligonucleotides are synthesized in accordance withthe Model 381A User Manual (Applied Biosystems). Sequence primers areused directly without any further purification. Other oligonucleotidesare purified up to a chain length of 70 by the OPC method(OPC=Oligonucleotide purification column, Applied Biosystems, ProductBulletin, January 1988). Longer oligonucleotides are purified bypolyacrylamide gel electrophoresis (6% acrylamide, 0.15% bisacrylamide,6 M urea, and TBE buffer) and after elution from the gel, desalinatedover a G-25 sepharose column.

EXAMPLE 7

Preparation of TNF-BP-Specific Hybridization Probes

The oligonucleotides were selected, with a view to using them to amplifycDNA by PCR:

-   a) From the N-terminal amino acid sequence of the TNF-binding    protein (main sequence, obtained from preliminary test 3 and Example    5, fraction 1)

Asp-Ser-Val-Cys-Pro-GLn-Gly-Lys-Tyr-Ile-His- Pro-GLn-a heptapeptide region was selected which permits the lowest possiblecomplexity of a mixed oligonucleotide for hybridizing to cDNA: these areamino acids 6 to 12. In order to reduce the complexity of the mixedoligonucleotide, four mixed oligonucleotides were prepared each having acomplexity of 48. The oligonucleotides were prepared in the direction ofthe mRNA and are thus oriented towards the 3′ end of the sequence andare identical to the non-coding strand of the TNF-BP gene:

Gln-Gly-Lys-Tyr-Ile-His-Pro 5′CAA GGT AAA TAT ATT CAT CC 3′TNF-BP #3/1EBI-     G       G   C   C   C 1639                     A 5′CAA GGC AAATAT ATT CAT CC 3′TNF-BP #3/2 EBI-     G       G   C   C   C 1640                    A 5′CAA GGA AAA TAT ATT CAT CC 3′TNF-BP #3/3 EBI-    G       G   C   C   C 1641                     A 5′CAA GGG AAA TATATT CAT CC 3′TNF-BP #3/4 EBI-     G       G   C   C   C 1642                    A

-   b) From the amino acid sequence of a tryptic peptide (fraction 11 of    the tryptic digestion) of the amino acid sequence

Glu-Cys-Glu-Ser-Gly-Ser-Phe-Thr-Ala-Ser-(Glu/Cys)-ASn-Asn-Lysa peptide region was selected and another set of mixed oligonucleotideswas synthesized:

-Phe-Thr-Ala-Ser-Glu-Asn-Asn-Lys                  Cys TNF-BP #4/5(EBI-1653): 3′AAA TGA CGG AGA CTC TTG TTG TT CCTAGGG 5′    G   G   T   T   T         T TNF-BP #4/6(EBI-1654): 3′AAA TGA CGG TCACTC TTG TTG TT CCTAGGG 5′     G   G   T   T   T         T TNF-BP#4/7(EBI-1657): 3′AAA TGA CGG AGA ACA TTG TTG TT CCTAGGG 5′    G   G   T   T   T         T TNF-BP #4/8(EBI-1658): 3′AAA TGA CGG TCAACA TTG TTG TT CCTAGGG 5′     G   G   T   T   T         T

The oligonucleotides were synthesized complementarily to mRNA and arethus oriented towards the 5′ end of the sequence. In order to allowefficient cloning of the amplified DNA fragment following PCR, a BamHIlinker was also provided at the 5′ end of the oligonucleotides. If, forexample, oligonucleotides TNF-BP Nos. 4/5–8 together with TNF-BP Nos.3/1–4 are used for PCR on the entire λ DNA of a library, any DNAfragment which results can be subsequently cut with BamHI. The partneroligonucleotides yield a straight end at the 5′ terminus andconsequently the fragment can be cloned into the SmaI-BamHl sites of asuitable vector.

Each mixed oligonucleotide TNF-BP No. 4/5 to 8 is a mixture of 48individual nucleotides and does not take into account a few codons,namely:

Thr ACG Ala GCG and GCT Ser TCG and TCC Asn AAT

In the case of GCT, the possibility that the triplet CGG complementaryto GCC (Ala) can be effective by forming a G-T bridge is taken intoconsideration, while in the case of TCG (Ser) and AAT (Asn), the sameapplies with regard to AGT and TTG, respectively.

ACG, GCG, and TCG are extremely rare codons (CG rule) and are thereforenot taken into consideration.

EXAMPLE 8

Amplification of a Partial Sequence Coding for TNF-BP from a cDNALibrary

a) Isolation of λ-DNA of a cDNA Library

The cDNA library was prepared using the method described in EP-A1 0 293567 for the human placental cDNA, with the difference that the startingmaterial used was 10⁹ fibrosarcoma cells of the cell line HS 913 T,which had been grown by stimulation with human TNF-α (10 ng/ml). Insteadof λ gt10, λ gt11 was used (cDNA synthesis: Amersham RPN 1256; EcoRIdigested λ gt11 arms: Promega Biotech; in vitro packaging of the ligatedDNA: Gigapack Plus, Stratagene).

5 ml of the phage supernatant of the amplified cDNA library of the humanfibrosarcoma cell line HS913T in 11 was mixed with 0.5 μg of RNase A and0.5 μg of DNase I and incubated for 1 hour at 37° C. The mixture wascentrifuged for 10 minutes at 5000×g, the supernatant was freed fromprotein by extraction with phenol and chloroform, and the DNA wasprecipitated from the aqueous phase by the addition of ethanol. The λDNA was dissolved in TE buffer (10 mM Tris, pH 7.5, 1 mM EDTA).

b) PCR Amplification of a TNF-BP Sequence from a cDNA Library

For the application of PCR (Saiki et al., Science 239:487–491 (1988)) toDNA from the HS913T cDNA library, 16 individual reactions were carriedout, in each of which one of the 4 mixed oligonucleotides EBI-1639,EBI-1640, EBI-1641, and EBI-1642 was used as the first primer and one ofthe four mixed oligonucleotides EBI-1653, EBI-1654, EBI-1657, andEBI-1658 was used as the second primer. Each of these mixedoligonucleotides contains 48 different oligonucleotides of equal length.

Amplification by means of PCR took place in 50 μl reaction volume,containing 250 ng of λ DNA from the cDNA library, 50 mM KCl, 10 mM Tris,pH 8.3, 1.5 mM MgCl₂, 0.01% gelatine, 0.2 mM of each of the 4deoxynucleoside triphosphates (DATP, dGTP, dCTP, and dTTP), 200 pmol ofeach of the first and second primers and 1.25 units of Taq polymerase(Perkin-Elmer Cetus). To prevent evaporation, the solution was coatedwith a few drops of mineral oil (0.1 ml); the PCR was carried out in aDNA Thermal Cycler (Perkin-Elmer Cetus) as follows: the samples wereheated to 94° C. for 5 minutes in order to denature the DNA, and thensubjected to 40 amplification cycles. One cycle consisted of 40 secondsincubation at 94° C., 2 minutes incubation at 55° C., and 3 minutesincubation at 72° C. At the end of the last cycle, the samples wereincubated at 72° C. for a further 7 minutes to ensure that the lastprimer lengthening had been completed. After cooling to ambienttemperature, the samples were freed from protein with phenol andchloroform, and the DNA was precipitated with ethanol.

5 μl of each of the 16 PCR samples were applied to an agarose gel andthe length of the amplified DNA fragments was determined afterelectrophoretic separation. The most intense DNA band, a fragment 0.16kb long, could be seen in the PCR samples which had been amplified withthe oligonucleotide EBI-1653 as the first primer and one of theoligonucleotides EBI-1639, EBI-1640, EBI-1641, or EBI-1642 as the secondprimer. Since the sample amplified with the pair of primers EBI-1653 andEBI-1642 contained the largest amount of this 0.16 kb DNA fragment, thissample was selected for further processing.

EXAMPLE 9

Cloning and Sequencing of a DNA Fragment Obtained by PCR Amplification

The PCR product of primers EBI-1642 and EBI-1653 obtained was cut withBamHI and subsequently separated by electrophoresis in an agarose gel(1.5% NuSieve GTG agarose plus 1% Seakem GTG agarose; FMC Corporation)according to size. The main band, a DNA fragment 0.16 kb long, waselectroeluted from the gel and precipitated with ethanol. This DNAfragment was ligated with BamHI/SmaI cut plasmid pUC18 (Pharmacia) andE. coli JMIO1 was transformed with the ligation mixture. The plasmidsprepared by the mini-preparation method were characterized by cuttingwith the restriction enzymes PvuII and EcoRI-BamHI and subsequentelectrophoresis in agarose gels. The plasmid pUC18 contains two cuttingsites for PvuII which flank the polycloning site in a 0.32 kb DNAfragment. Very short DNA inserts in the polycloning site of the plasmidcan be made visible more easily in agarose gel after cutting with PvuIIsince the length is extended by 0.32 kb. By cutting with EcoRI andBamHI, the DNA fragment ligated into the plasmid vector cut with BamHIand SmaI, including some base pairs of the polylinker sequence, can beobtained. A clone with the desired insert has been designated pTNF-BP3B.The entire DNA insert of this clone was sequenced after subcloning of anEcoRI-BamHI fragment in M13 mp18 (Pharmacia) by the modified dideoxymethod using sequenase (United States Biochemical Corporation).

Analysis of the PCR-amplified DNA gave the following sequence (only thenon-coding strand is shown, and above it the derived amino acidsequence):

                  5                  10 Gln Gly Lys Tyr Ile His Pro GlnAsn Asn Ser Ile Cys CAG GGG AAA TAT ATT CAC CCT CAA AAT AAT TCG ATT TGC    15                  20                  25 Cys Thr Lys Cys His LysGly Thr Tyr Leu Tyr Asn Asp TGT ACC AAG TGC CAC AAA GGA ACC TAC TTG TACAAT GAC             30                  35 Cys Pro Gly Pro Gly Gln AspThr Asp Cys Arg Glu Cys TGT CCA GGC CCG GGG CAG GAT ACG GAC TGC AGG GAGTGT 40                  45                  50 Glu Ser Gly Ser Phe ThrAla Ser Glu Asn Asn Lys GAG AGC GGC TCC TTC ACA GCC TCA GAA AAC AAC AAGGAT CC

The first 20 and last 29 nucleotides (underlined script) correspond tothe sequences of the primer oligonucleotides EBI-1642 and the complementof EBI-1653, respectively. Amino acids 38 to 43 confirm the remainingsequence of the tryptic peptide 11. Furthermore, the DNA fragmentproduced by PCR contains the sequence of the peptide of fraction 20 ofthe tryptic digestion (amino acids 20 to 34, underlined). This showsthat the clone pTNF-BP3B was derived from a cDNA which codes for TNFbinding protein. pTNF-BP3B therefore constitutes a probe, e.g., forsearching for TNF-BP cDNAs in cDNA libraries.

EXAMPLE 10

a. Isolation of TNF-BP cDNA Clones

About 720,000 phages of the HS913T cDNA library in λ gt11 were plated onE. coli Y1088 (ΔlacU169, pro::Tn5, tonA2, hsdR, supE, supF, metB, trpR,F-, λ-, (pMC9)) (about 60,000 phages per 14.5 cm petri dish, LB-agar: 10g/l tryptone, 5 g/l of yeast extract, 5 g/l of NaCl, 1.5% agar, platingin top agarose: 10 g/l of tryptone, 8 g/l of NaCl, 0.8% agarose). Twonitrocellulose filter extracts were prepared from each plate. Thefilters were prewashed (16 hours at 65° C.) in:

-   -   50 mM Tris-HCl, pH 8.0    -   1 M NaCl    -   1 mM EDTA    -   0.1% SDS        The filters were pre-hybridized for two hours at 65° C. in:    -   6×SSC (0.9M NaCl, 0.09 M trisodium citrate)    -   5× Denhardt's (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1%        BSA=bovine serum albumin)    -   0.1% SDS

Preparation of the radioactively labeled probe: pTNF-BP 3B was doublycut with BamHI and EcoRI and the approximately 0.16 kb insert wasisolated. 0.6 μg of the insert in 32 μl was denatured at 100° C. andprimed with 60 pmol each of EBI-1642 and EBI-1653 by cooling to 80° C.over 10 minutes and rapid cooling in ice water.

After the addition of:

-   -   10 μl α-³²P-dCTP (100 μCi, 3.7 MBq)    -   5 μl 10× priming buffer (0.1 M Tris-HCl, pH 8.0, 50 mM MgCl₂)    -   2 μl 1 mM DATP, dGTP, and dTTP    -   1 μl PolIK (Klenow fragment of E. coli DNA polymerase I, 5        units)        Incubation was carried out for 90 minutes at ambient        temperature. After heat inactivation (10 minutes at 70° C.), the        non-incorporated radioactivity was removed by chromatography on        Biogel P6DG (Biorad) in TE buffer (10 mM Tris-HCl, pH 8, 1 mM        EDTA). 65×10⁶ cpm were incorporated. The hybridization of the        filters was carried out in a total volume of 80 ml of 6×SSC/5×        Denhardt's/0.1% SDS plus heat-denatured hybridizing probe for 16        hours at 65° C. The filters were washed twice for 30 minutes at        ambient temperature in 6×SSC/0.01% SDS and once for 45 minutes        at ambient temperature in 2×SSC/0.01% SDS and three times for 30        minutes at 65° C. in 2×SSC/0.01% SDS. The filters were air dried        and then exposed to Amersham Hyperfilm for 16 hours using an        intensifier screen at −70° C. In all, 30 hybridizing plaques        were identified (λ-TNF-BP Nos. 1–30). The regions with the        hybridizing plaques were pricked out as precisely as possible        and the phages were eluted in 300 μl of SM buffer plus 30 μl of        chloroform. By plaque purification (plating of about 200 phages        per 9 cm petri dish on the second passage, or about 20 phages        per 9 cm petri dish on the third passage, filter extracts        doubled, preparation, hybridization, and washing as described in        the first search) 25 hybridizing phages were finally separated        (λ-TNF-BP #1–10, 12–24, 29, and 30).        b. Preparation of the Recombinant λ DNA from the Clones λ-TNF-BP        Nos. 13, 15, 23, and 30

2×10⁶ phages were plated on E. coli Y1088 in top agarose (10 g/ltryptone, 8 g/l NaCl 0.8% agarose) (14.5 cm petri dish) with LB agarose(1.5% agarose, 0.2% glucose, 10 mM MgSO₄, 10 g/l tryptone, 5 g/l yeastextract, 5 g/l NaCl) and incubated at 37° C. for 6 hours. After theplates had been cooled (30 minutes at 4° C.) they were coated with 10 mlof λdiluent (10 mM Tris-HCl, pH 8.0, 10 mM MgCl₂, 0.1 mM EDTA) andeluted for 16 hours at 4° C. The supernatant was transferred into 15 mlCorex test tubes and centrifuged for 10 minutes at 15,000 rpm and at 4°C. (Beckman J2–21 centrifuge, JA20 rotor). The supernatant was decantedinto 10 ml polycarbonate test tubes and centrifuged at 50,000 rpm at 20°C. until (ω²t=3×10¹⁰ (Beckman L8–70, 50 Ti rotor). The pellet wasresuspended in 0.5 ml of λ diluent and transferred into Eppendorf testtubes (1.4 ml). After the addition of 5 μg of RNase A and 0.5 μg DNaseIand incubation at 37° C. for 30 minutes and the addition of 25 μl of 0.5M EDTA, 12.5 μl of 1 M Tris-HCl, pH 8.0, and 6.5 μl of 20% SDS,incubation was continued at 70° C. for 30 minutes. The λ DNA waspurified by phenol/chloroform extraction and precipitated with ethanol.Finally, the DNA was dissolved in 100 μl of TE buffer.

EXAMPLE 11

Subcloning and Sequencing of TNF-BP cDNA Clones 15 and 23

In order to characterize the cDNAs of the clones λ-TNF-BP15 andλ-TNF-BP23, which showed the strongest signals during hybridization, thecDNA inserts were cut out of the λ DNA with EcoRI, then afterelectrophoretic separation were eluted from an agarose gel andprecipitated with ethanol. The DNA fragments of 1.3 kb (from λ-TNF-BP15)and 1.1 kb (from λ-TNF-BP23) were cut with EcoRI, ligated with T4 DNAligase to the plasmid vector pT7/T3α-18 (Bethesda ResearchLaboratories), which was dephosphorylated with calf intestine alkalinephosphatase, and E. coli JM101 was transformed. From individual coloniesof bacteria which showed no blue staining after selection on agaroseplates with ampicillin and X-gal, plasmid DNA was prepared in amini-preparation process and the presence and orientation of the cDNAinsert was determined by cutting with EcoRl and HindIII. Plasmids whichcontained the EcoRl insert of the phages λ-TNF-BP15 or λ-TNF-BP23,oriented in such a way that the end corresponding to the 5′-end of themRNA was facing the T7 promotor, were designated pTNF-BP15 andpTNF-BP23, respectively. The EcoRI inserts of λ-TNF-BP15 and λ-TNF-BP23were also ligated to the M13 mp19 vector which had been cut with EcoRIand dephosphorylated, and E. coli JM101 was transformed. From a fewrandomly selected M13 clones, single-stranded DNA was prepared and usedas the basis for sequencing by the dideoxy method. On M13 clones whichcontained the cDNA inserts in the opposite orientation, both DNA strandswere fully sequenced using the universal sequencing primer andspecifically synthesized oligonucleotide primers which bind to the cDNAinsert.

The complete nucleotide sequence of 1334 bases of the cDNA insert ofλ-TNF-BP15, or pTNF-BP15, is shown in FIGS. 1A–1C. Bases 1–6 and1328–1334 correspond to the EcoRI linkers which had been added to thecDNA during the preparation of the cDNA library. The nucleotide sequenceof the cDNA insert of λ-TNF-BP23 corresponds to that of λ-TNF-BP15(bases 22–1100), flanked by EcoRI linkers.

The clone λ-TNF-BP30 was also investigated; its sequence corresponds toλ-TNF-BP15, except that the sequence has a deletion of 74 bp (nucleotide764 to 837).

EXAMPLE 12

Construction of the Expression Plasmid pAD-CMV1 and pAD-CMV2

From parts of the expression plasmids pCDM8 (Seed and Aruffo, Proc.Natl. Acad. Sci. 84:8573–8577 (1987); Seed, B. Nature 329:840–842(1987)); Invitrogen), pSV2gptDHFR20 (EP-A1 0 321 842) and the plasmidBluescript SK+ (Short, J. M., et al., Nucl. Acids Res. 11:5521–5540(1988); Stratagene) a new plasmid was constructed which has amulticloning site for the directed insertion of heterologous DNAsequences and which can be replicated in E. coli by means of ampicillinresistance with a high copy number. The intergenic region of M13 makesit possible to produce single-stranded plasmid DNA by superinfection ofthe transformed bacteria with a helper phage (e.g., R408 or M13K07) tofacilitate sequencing and mutagenesis of the plasmid DNA. The T7promotor which precedes the multicloning site makes it possible toprepare RNA transcripts in vitro. In mammalian cells, the expression ofheterologous genes is driven by the cytomegalovirus (CMV)promotor/enhancer (Boshart, M., et al., Cell 41:521–530 (1985)). TheSV40 replication origin makes it possible, in suitable cell lines (e.g.,SV40 transformed cells such as COS-7, adenovirus transformed cell line293 (ATCC CRL1573)), to carry out autonomous replication of theexpression plasmid at high copy numbers and thus at high rates intransient expression. For preparing permanently transformed cell linesand subsequently amplifying the expression cassette by means ofmethotrexate, a modified hamster minigene (promotor with coding regionand the first intron) for dihydrofolate reductase (DHFR) is used as theselection marker.

A) Preparation of the Vector and Promotor Sections by PCR

The plasmid Bluescript SK+ was linearized with HindIII and 5 ng of DNAwas used in a 100 μl PCR mixture (reaction buffer: 50 mM KCl, 10 mMTris-HCl, pH 8.3, 1.5 mM MgCl₂, 0.01% (w/v) gelatine, 0.2 mM of the fourdeoxynucleotide triphosphates (dATP, dGTP, dCTP, and dTTP), 2.5 units ofTaq polymerase per 100 μl. The primers used were 50 pmol of thesynthetic oligonucleotides EBI-1786 (5′-GGAATTCAGCCTGAATGGCGAATGGG-3′;SEQ ID NO: 80; binds just outside the M13 ori-region at Bluescriptposition 475, independently of the M13 ori-orientation) and EBI-1729(5′-CCTCGAGCGTTGCTGGCGTTTTTCC-3′; SEQ ID NO: 81; binds to Bluescript atposition 1195 in front of ori; corresponds to the start of theBluescript sequence in pCDM8; 6 bases 5′ yield XhoI). After 5 minutesdenaturation at 94° C., PCR was carried out over 20 cycles (40 secondsat 94° C., 45 seconds at 55° C., 5 minutes at 72° C.; Perkin-Elmer CetusThermal Cycler). The oligonucleotides flank the intergenic region of M13or the replication origin (ori) with the intermediate gene forβ-lactamase. At the same time, at the end of the replication origin anXhoI cutting site is produced and at the other end an EcoRI cutting siteis produced. The reaction mixture was freed from protein by extractionwith phenol/chloroform and the DNA was precipitated with ethanol. TheDNA obtained was cut with XhoI and EcoRI, and after electrophoresis inan agarose gel, a fragment of 2.3 kb was isolated.

5 ng of plasmid pCDM8 linearized with SacII was amplified by PCR withthe oliqonucleotides EBI-1733 (5′-GGTCGACATTGATTATTG-ACTAG-3′; SEQ IDNO: 82; binds to CMV promotor region (position 1542) of pCDM8,corresponding to position 1 in pAD-CMV; SalI site for cloning) andEBI-1734 (5′-GGAATTCCCTAGGAATACAGCGG-3′; SEQ ID NO: 83; binds to polyomaorigin of 3′SV40 polyA region in pCDM8 (position 3590)) under identicalconditions to those described for Bluescript SK+. The oligonucleotidesbind at the beginning of the CMV promotor/enhancer sequence and producea SalI cutting site (EBI-1733) or bind to the end of the SV40poly-adenylation site and produce an EcoRI cutting site (EBI-1734). ThePCR product was cut with SalI and EcoRI and a DNA fragment of 1.8 kb wasisolated from an agarose gel.

The two PCR products were ligated with T4 DNA ligase, E. coli HB101 wastransformed with the resulting ligation product, and plasmid DNA wasamplified and prepared using standard methods. The plasmid of thedesired nature (see FIGS. 3A–3B) was designated pCMV-M13. The SV40replication origin (SV40 ori) was isolated from the plasmidpSV2gptDHFR20 (EP-A1 0 321 842). To do this, this plasmid was doubledigested with HindIII and PvuII and the DNA ends were blunted bysubsequent treatment with the large fragment of E. coli DNA polymerase(Klenow enzyme) in the presence of the four deoxynucleotidetriphosphates. A 0.36 kb DNA fragment thus obtained was isolated from anagarose gel and ligated into pCMV-M13, which was linearized with EcoRI.A plasmid obtained after transformation of E. coli HB101, with the SV40ori in the same orientation as the β-lactamase gene and the CMVpromotor, was designated pCMV-SV40. The construction of this plasmid isshown in FIGS. 3A–3B.

b) Mutagenesis of the DHFR Gene

In order to prepare an expression plasmid with a versatile multicloningsite, two restriction enzyme cutting sites were removed from the DHFRminigene by directed mutagenesis and three such sites were removed bydeletion. To do this, a 1.7 kb BgIII fragment from the plasmidpSV2gptDHFR20, containing the entire coding region of the hamster DHFRgene, was cloned into the BgIII site of the plasmid pUC219 (IBI) and theplasmid pUCDHFR was obtained. E. coli JM109 (Stratagene) cellstransformed with pUCDHFR were infected with an approximately 40-foldexcess of the helper phage R408 (Stratagene) and shaken in LB medium for16 hours at 37° C. Single-stranded plasmid DNA was isolated from thebacterial supernatant.

Controlled mutagenesis was carried out in two successive steps, usingthe in vitro mutagenesis system RPN1523 (Amersham) The EcoRI sitelocated at the beginning of Exon 2 was destroyed by exchanging a basefrom GAATTC to GAGTTC. This base exchange does not result in any changein the coded amino acid sequence and furthermore corresponds to thenucleotide sequence in the natural murine DHFR gene (McGrogan, M., etal., J. Biol. Chem. 260:2307–2314 (1985); Mitchell, P. J., et al., Mol.Cell. Biol. 6:425–440 (1986)). An oligonucleotide (antisenseorientation) of the sequence 5′-GTACTTGAACTCGTTCCTG-3′ (SEQ ID NO: 84;EBI-1751) was used for the mutagenesis. A plasmid with the desiredmutation was prepared as single strand DNA as described above and thePstI site located in the first intron was removed by mutagenesis withthe oligonucleotide EBI-1857 (antisense orientation;5′-GGCAAGGGCAGCAGCCGG-3′; SEQ ID NO: 85) from CTGCAG into CTGCTG. Themutations were confirmed by sequencing and the resulting plasmid wasdesignated pUCDHFR-Mut2.

The 1.7 kb BglII fragment was isolated from the plasmid pUCDHFR-Mut2 andligated into plasmid pSV2gptDHFR20, double digested with BglII andBamHI. After transformation of E. coli, amplification, and DNAisolation, a plasmid of the desired nature was obtained, which wasdesignated pSV2gptDHFR-Mut2. By cutting with BamHI, in the 3′-non-codingregion of the DHFR gene, a 0.12 kb DNA fragment following the BglII sitewas removed, which also contains a KpnI cutting site. By linking theoverhanging DNA ends formed with BglII and BamHI, the recognitionsequences for these two enzymes were also destroyed.

The plasmid pCMV-SV40 was double digested with EcoRI and BamHI and theDNA ends were then blunted with Klenow enzyme. The DNA was purified byextraction with phenol/chloroform and ethanol precipitation, thendephosphorylated by incubation with alkaline phosphatase, and the 4.4 kblong vector DNA was isolated from an agarose gel.

The plasmid pSV2gptDHFR-Mut2 (FIGS. 4A–4B) was double digested withEcoRI and PstI and the DNA ends were blunted by 20 minutes incubation at11° C. with 5 units of T4 DNA polymerase (50 mM Tris-HCl, pH 8.0, 5 mMMgCl₂, 5 mM dithiothreitol, 0.1 mM of each of the four deoxynucleotidetriphosphates, and 50 μg/ml of bovine serum albumin). The 2.4 kb longDNA fragment with the mutated DHFR gene was isolated from an agarose geland ligated with the pCMV-SV40 prepared as described above. A plasmidobtained after transformation of E. coli and containing the DHFR gene inthe same orientation as the CMV promotor was designated pCMV-SV40DHFR.

In the last step, the 0.4 kb stuffer fragment after the CMV promotor,which originated from the original plasmid pCDM8, was exchanged for amulticloning site. To do this, the plasmid pCMV-SV40DHFR was doubledigested with HindIII and XbaI and the vector part was isolated from anagarose gel. The multicloning site formed from the two oligonucleotidesEBI-1823 (5′-AGCTTCTGCAGGTC-GACATCGATGGATCCGGTACCTCGAGCGGCCGCGAATTCT-3′;SEQ ID NO: 86) and EBI-1829(5′-CTAGAGAATTCGCGGCCGCTCGAGGTACCGGATCCATCGATGTCGACCTGC-AGA-3′; SEQ IDNO: 87) contains (including the ends which are compatible for cloning inHindIII-XbaI) restriction cutting sites for the enzymes PstI, SalI,ClaI, BamHI, KpnI, XhoI, NotI, and EcoRI.

1 μg of each of the two oligonucleotides was incubated for 1 hour at 37°C. in 20 μl of reaction buffer (70 mM Tris-HCl, pH 7.6, 10 mM MgCl₂, 5mM dithiothreitol, 0.1 mM ATP) with 5 units of T4 polynucleotide kinasein order to phosphorylate the 5′ ends. The reaction was stopped byheating to 70° C. for 10 minutes and the complementary oligonucleotideswere hybridized with one another by incubating the sample for a further10 minutes at 56° C. and then slowly cooling it to ambient temperature.4 μl of the hybridized oligonucleotides (100 ng) were ligated with about100 ng of plasmid vector and E. coli HB101 was transformed. A plasmidwhich was capable of being linearized with the enzymes of themulticloning site (with the exception of NotI) was designed pAD-CMV1. Ofa number of clones tested, it was not possible to identify any one theplasmids which could be cut with NotI. Sequencing always showed thedeletion of some bases within the NotI recognition sequence.

In the same way, the expression plasmid pAD-CMV2, which contains therestriction cutting sites within the multicloning site in the reverseorder, was obtained with the oligonucleotide pair EBI-1820(5′-AGCTCTAGAGAATTCGCGGCCGCTCGAGGTACCGGATCCATCGATG-TCGACCTGCAGAAGCTTG-3′;SEQ ID NO: 88) and EBI-1821(5′-CTAGCAAGCTTCTGCAGGTCGACATCGATGGATCCGGTACCTCGAGCGGCCGCGAATTCTCTAG-3′;SEQ ID NO: 89). The plasmid pAD-CMV2 was obtained which was capable ofbeing linearized with all the restriction enzymes, including NotI.

The nucleotide sequence of the 6414 bp plasmid pAD-CMV1 (FIG. 5B) isshown in full in FIGS. 6A–6E.

The sections of the plasmid (specified in the numbering of the bases)correspond to the following sequences:

-   -   1–21 EBI-1733, beginning of CMV enhancer-promotor (from CDM8)    -   632–649 T7 promotor    -   658–713 Multicloning site (HindIII to XbaI from EBI-1823,        EBI-1829)    -   714–1412 SV40 intron and poly-adenylation site (from CDM8)    -   1413–2310 5′-non-coding region and promotor of the hamster DHFR        gene (from pSV2gptDHFR20)    -   2311–2396 Hamster DHFR: Exon I    -   2516 A to T mutation destroys PstI site in DHFR intron 1    -   2701–3178 DHFR Exons 2–6 (coding region)    -   2707 A to G mutation destroys EcoRI site    -   3272–3273 Deletion between BglII and BamHI in DHFR 3′ non-coding        region    -   3831 End of DHFR gene (from pSV2gptDHFR20)    -   3832–4169 SV40 ori (from pSV2gptDHFR20)    -   4170–4648 M13 ori (from pBluescript SK+)    -   4780–5640 β-lactamase (coding region)    -   6395–6414 EBI-1729, end of the pBluescript vector sequence

The preparation of the plasmids pAD-CMV1 and pAD-CMV2 is shown in FIGS.5A–5B.

EXAMPLE 13

Construction of the Plasmid pADTNF-BP for the Expression of the SolubleForm of TNF-BP

In order to prepare the secreted form of TNF-BP by the direct method, atranslation stop codon was inserted in the cDNA coding for part of theTNF receptor (see Example 11; hereinafter designated TNF-R cDNA) afterthe codon of the C-terminal amino acid of the natural TNF-BP (AAT,Asn-172; corresponding to position 201 in FIG. 9A). In this way theprotein synthesis is broken off at this point, making it possible tosecrete TNF-BP directly into the cell supernatant without having toundergo a subsequent reaction, which might possibly be rate determiningof proteolytic cleaving of sections of the TNF receptor located in theC-terminal direction.

At the same time as the stop codon was inserted by PCR, the5′-non-coding region of the TNF-R cDNA was shortened in order to removethe translation start codon of another open reading frame (bases 72–203in FIG. 9A), which is located 5′ from that of the TNF-R, and a BamHI orEcoRI cutting site is inserted at the 5′ or 3′-end of the cDNA.

100 ng of plasmid pTNF-BP15 linearized with XmnI (see Example 11) wasamplified with 50 pmol of the oligonucleotides EBI-1986 (sense;5′-CAGGATCCGAGTCTCAACCCTCAAC-3′; SEQ ID NO: 90) and EBI-1929 (antisense;5′-GGGAATTCCTTATCAATTCTCAATCTGGGGTAGGCACAACTTC-3′; SEQ ID NO: 91;insertion of two stop codons and an EcoRI site) in a 100 μl PCR mixtureover 10 cycles. The cycle conditions were 40 minutes at 94° C., 45seconds at 55° C., and 5 minutes at 72° C. After the last cycle,incubation was continued for a further 7 minutes at 72° C. and thereaction was stopped by extracting with phenol/chloroform. The DNA wasprecipitated with ethanol and then double digested with BamHI and EcoRI.The resulting 0.75 kb DNA fragment was isolated from an agarose gel andcloned into plasmid pT7/T3α-19 (BRL) double digested with BamHI andEcoRI. One of the plasmids obtained, which was found to have the desiredsequence when the entire insert was sequenced, was designated pTNF-BP.

pTNF-BP was cut with BamHI and EcoRI and the 0.75 kb DNA insert wascloned into the expression plasmid pAD-CMV1 cut with BamHI and EcoRI. Aplasmid obtained with the desired composition was designated PADTNF-BP(FIG. 7A).

EXAMPLE 14

Construction of the Plasmid pADBTNF-BP for the Expression of the Solubleform of TNF-BP

For another variant of an expression plasmid for the production ofsecreted TNF-BP, the 5′-non-coding region of TNF-R cDNA was exchangedfor the 5′-non-coding region of human β-globin mRNA. The reason for thiswas the finding that the nucleotide sequence immediately before thetranslation start codon of the TNF-R sequence differs significantly fromthe concensus sequence found for efficient expression of eukaryoticgenes (Kozak, 1987), whereas the 5′-non-coding region of the β-globinmRNA corresponds extremely well to this concensus sequence (Lawn et al.,1980). By means of the oligonucleotide EBI-2452(5′-CACAGTCGACTTACATTTG-CTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGGCCTCTCCACCGTGC-3′;SEQ ID NO: 92), which contained after a SalI restriction cutting sitethe authentic 5′-non-coding sequence corresponding to the human β-globinmRNA sequence followed by 20 bases of the coding region of TNF-BP, theTNF-R sequence was modified by PCR. 100 ng of plasmid pTNF-BP linearizedwith EcoRI was amplified in 100 μl of reaction mixture with 50 pmol eachof the oligonucleotides EBI-2452 and EBI-1922 (antisense;5′-GAGGCTGCA-ATTGAAGC3′; SEQ ID NO: 93; binds to the huTNF-R sequence atposition 656) for 20 cycles (40 seconds at 94° C., 45 seconds at 55° C.,90 seconds at 72° C.). After the PCR product was purified by extractionwith phenol/chloroform and ethanol precipitation, the DNA was doubledigested with SalI and BglII and the resulting 0.51 kb DNA fragment wasisolated from an agarose gel. The corresponding part of the TNF-Rsequence was removed from the plasmid pTNF-BP by cutting with SalI andBglII, and the 3.1 kb long plasmid portion was isolated from an agarosegel and ligated with the 0.51 kb long PCR product. After transformationof E. coli, seven of the resulting plasmids were sequenced. One of theseplasmids contained precisely the desired sequence. This plasmid wasdesignated pBTNF-BP. The entire SalI-EcoRl insert of pBTNF-BP was clonedinto the similarly cut expression plasmid pAD-CMVI and the resultingplasmid was designated pADBTNF-BP (FIG. 7B).

EXAMPLE 15

Isolation of Rat TNF-R cDNA Clones

First of all, rat brain cDNA was prepared analogously to the HS913T cDNAlibrary (see Example 4) from the rat Glia tumour cells lines C6 (ATCCNo. CCL107) in λ gt11.

600,000 phages of the rat brain cDNA library in λ gt11 were screened byhybridization as described in Example 6. The probe used was the purifiedEcoRI insert of pTNF-BP30 (cf. Example 6). About 100 ng of DNA wasradioactively labeled with 1 μg of random hexamer primer instead of thespecific oligonucleotides as described in Example 6, using [α-³²P]dCTP.25×10⁶ cpm were incorporated. Hybridization of the filters was carriedout under the same conditions as in Example 6. The filters were washedtwice for 30 minutes at ambient temperature in 2×SSC/0.1% SDS and threetimes for 30 minutes at 65° C. in 2×SSC/0.1 SDS and twice for 30 minutesat 65° C. in 0.5×SSC/0.5% SDS. The air dried filters were then exposedto Kodak XAR X-ray film for 16 hours using an intensifier film at −70°C. A total of 10 hybridizing plaques were identified and separated byplaque purification. After plaque purification had been carried outthree times, three λ clones (λ-raTNF-R Nos. 3, 4, and 8) were finallyseparated out and the phage DNA was prepared as described.

The length of the cDNA insert was determined, after cutting the λ DNAwith EcoRI and separation in an agarose gel, to be 2.2 kb for the clonesraTNF-R3 and raTNF-R8 and 2.1 kb for the clone raTNF-R4. The EcoRIinserts of the clones λ-raTNF-R3 and λ-raTNF-R8 were cloned intosimilarly cut M13 mp19, and the DNA sequence was determined withuniversal sequencing primers and specifically synthesizedoligonucleotide primers.

The complete nucleotide sequence of raTNF-R8 is shown in FIGS. 8A–8B.The first and last seven bases correspond to the EcoRI linkers which hadbeen added during the preparation of the cDNA library.

EXAMPLE 16

Isolation of a Clone Containing the Complete cDNA Coding for the HumanTNF Receptor

The complete cDNA of the rat TNF-R made it easier to search for themissing 3′ part of human TNF-R cDNA. The probe used for thehybridization was the 0.4 kb long PCR product of the primers EBI-2316(5′-ATTCGTGCGGCGCCTAG-3′; SEQ ID NO: 94; binds to TNF-R with the 2ndbase of EcoRI, breaks off at the TNF-R cDNA) and EBI-2467(5′-GTCGGTAGCACCAAGGA-3′; SEQ ID NO: 95; binds about 400 bases beforepoly-A to cDNA clone, corresponds to position 1775 in raTNF-R) withλ-raTNF-R8 as the starting material. This DNA fragment corresponds tothe region of rat TNF-R cDNA which had been assumed to follow theinternal EcoRI site in human TNF-R.

2.5×10⁶ cpm of the raTNF-R probe were used to screen 600,000 plaques ofthe HS913T cDNA library. The hybridization conditions corresponded tothose specified in Example 10. The filters were washed twice for 30minutes at ambient temperature in 2×SSC/0.1% SDS and twice for 30minutes at 65° C. in 2×SSC/0.1% SDS, air dried, and exposed to Kodak XARX-ray film using an intensifier screen for a period of 3 days at −70° C.Six positive plaques were identified and purified to two further roundsof plaques and λ DNA was prepared (λ-TNF-R Nos. 2, 5, 6, 8, 11, and 12).After the λ DNA was cut with EcoRI, all the clones had a DNA band ofabout 0.8 kb long. λ-TNF-R2 and λ-TNF-R11 additionally contained anEcoRI fragment of 1.3 kb. The two EcoRI inserts from λ-TNF-R2 weresubcloned into the EcoRI site of plasmid pUC218 (IBI) and thensequenced. The sequence of the 1.3 kb EcoRI fragment corresponded tothat of cDNA clone pTNF-BP15, and the 0.8 kb EcoRI fragment correspondedto the 3′ part of TNF-R mRNA, and contains, in front of the EcoRI linkersequence, a poly-A tail with 16 A residues. λ-TNF-R2 therefore containsthe complete coding region for human TNF-R, shown in FIGS. 9A–9B.

EXAMPLE 17

Construction of the Plasmids pADTNF-R and pADBTNF-R for Expression ofthe Entire Human TNF Receptor

First of all, as described in Example 13 for pTNF-BP or pADTNF-BP, aplasmid was constructed in which the 5′-non-coding region of pTNF-BP15had been shortened, but, unlike the plasmids described in Example 13,the 3′-end of pTNF-BP15 was retained. For this purpose, under identicalconditions to those described in Example 13, pTNF-BP15 was amplified byPCR using the oligonucleotide EBI-1986 and the M13–40 universal primer(5′-GTTTTCCCAGTCACGAC-3′; SEQ ID NO: 96). The PCR product was doubledigested with BamHI and EcoRI and cloned into the plasmid pT7/T3α-19.One of the plasmids obtained was designated pTMF-BP15B.

pTNF-BP15B was cut with BamHI and EcoRI and the 1.26 kb DNA insert wascloned into expression plasmid pAD-CMV1 cut with BamHI and EcoRI. Aplasmid of the desired composition thus obtained was designatedpADTNF-BP15.

This plasmid was linearized with EcoRI and the 0.8 kb EcoRI fragmentisolated from λTNF-R2 was cloned into the cutting site. Aftertransformation of E. coli, a few randomly isolated plasmids were checkedby cutting with various restriction enzymes for the correct orientationof the EcoRI fragment used. A plasmid designated pADTNF-R (FIG. 7C) wasinvestigated further for correct orientation by sequencing the insert,starting from the 3′-end of the inserted cDNA, with the oligonucleotideEBI-2112 (5′GTCCAATTATGTCACACC-3′; SEQ ID NO: 97), which binds to theplasmid pAD-CMV1 and its derivatives after the multicloning site.

Another expression plasmid in which the 5′-non-coding region of theTNF-R is exchanged for that of β-globin was constructed. PlasmidpADBTNF-BP was cut completely in order to remove the 1.1 kb BglIIfragment, the DNA ends were then dephosphorylated with calf intestinalalkaline phosphatase, and the plasmid vector (5.9 kb) with the β-globin5′-non-coding region of the β-globin gene and the 5′ part of the TNF-Rcoding region was isolated from an agarose gel. Plasmid pADTNF-R was cutwith BglII and the 2.5 kb DNA fragment containing the 3′ section of theTNF-R cDNA, as far as the promotor region of the following DHFR gene,was isolated from an agarose gel, and cloned into the plasmid vectorwhich had been prepared beforehand. A plasmid obtained aftertransformation of E. coli, having the BglII fragment inserted in thecorrect orientation, was designated pADBTNF-R (FIG. 7D).

EXAMPLE 18

Expression of Soluble TNF-BP in Eukaryotic Cell Lines

a) ELISA Test

In this Example TNF-BP was detected by the ELISA test as follows:

Each well of a 96 well microtitre plate was coated with 50 μl of 1:3000diluted polyclonal rabbit serum (polyclonal rabbit antibodies; preparedby precipitation of antiserum with ammonium sulphate; finalconcentration 50% saturation) against natural TNF-BP for 18 hours at 4°C., washed once with 0.05% Tween 20 in PBS, and free binding sites wereblocked with 150 to 200 μl of 0.5% bovine serum albumin, 0.05% Tween 20in PBS (PBS/BSS/Tween) for one hour at ambient temperature. The wellswere washed once with 0.05% Tween 20 in PBS and 50 μl of cellsupernatant or known quantities of natural TNF-BP (see Tables 4 and 5)and 50 μl of a 1:10,000-fold dilution of a polyclonal mouse serumagainst TNF-BP was applied and incubated for two hours at ambienttemperature. Then the wells were washed three times with 0.05% Tween 20in PBS and 50 μl of rabbit anti-mouse Ig-peroxidase conjugate (DakoP161; 1:5000 in PBS/BSA/Tween) was added and incubation was continuedfor a further two hours at ambient temperature. The wells were washedthree times with Tween/PBS and the staining reaction was carried outwith orthophenylenediamine (3 mg/ml) and Naperborate (1 mg/ml) in 0.067M potassium citrate, pH 5.0, 100 μl per well, for 20 minutes at ambienttemperature away from the light. After the addition of 100 μl of 4NH₂SO₄, the color intensity at a wavelength of 492 nm was measuredphotometrically in a microfilm plate photometer.

B) Transient Expression of Soluble TNF-BP in Eukaryotic Cell Lines

About 10⁶ cells (COS-7) per 80 mm petri dish were mixed with 10%heat-inactivated fetal calf serum in RPMI-1640 medium 24 hours beforetransfection and incubated at 37° C. in a 5% CO₂ atmosphere. The cellswere separated from the petri dish using a rubber spatula, centrifugedfor 5 minutes at 1200 rpm at ambient temperature (Heraeus minifuge,swing-out rotor 3360), washed once with 5 ml of serum-free medium,centrifuged for 5 minutes at 1200 rpm, suspended in 1 ml of medium mixedwith 250 μg/ml of DEAE dextran and 10 μg of plasmid DNA (see Table 4),and purified by carrying out CsCl density gradient centrifugation twice.The cells were incubated for 40 minutes at 37° C., washed once with 5 mlof medium containing 10% calf serum, and suspended in 5 ml of mediumwith 100 μg/ml of chloroquin. The cells were incubated for one hour at37° C., washed once with medium, and incubated with 10 ml of freshmedium at 37° C. After 72 hours, the cell supernatant was harvested andused to detect the secreted TNF-BP.

TABLE 4 Cell line COS-7 without plasmid <5 ng/ml pADTNF-BP 7.5 ng/mlpADBTNF-BP 146 ng/mlC) Preparations of Cell Lines which Permanently Produce TNF-BP

The dihydrofolate reductase (DHFR)-deficient hamster ovarial cell lineCHO DUKX BII (Urlaub and Chasin, 1980) was transfected with the plasmidpADBTNF-BP by calcium phosphate precipitation (Current Protocols inMolecular Biology, 1987). Four thickly grown cell culture flasks (25cm², 5 ml of culture medium per flask) were transfected with 5 μg ofDNA; after four hours incubation at 37° C. the medium was removed andreplaced by 5 ml of selection medium (MEM alpha medium with 10% dialyzedfetal calf serum). After incubation overnight, the cells were detachedusing trypsin solution; the cells from each flask were divided betweentwo 96-well tissue culture plates (100 μl per well in selection medium).Fresh medium was added at about weekly intervals. After about fourweeks, cell clones could be observed in 79 wells. The supernatants weretested for TNF-BP activity by the ELISA test. 37 supernatants showedactivity in ELISA. The results of the ELISA test of some positive clonesare shown in Table 5.

TABLE 5 Sample Absorption at 492 nm TNF-BP Standard  1 ng/ml 0.390  10ng/ml 1.233 100 ng/ml 1.875 Culture medium (negative control) 0.085Clone AlG3 0.468 A2F5 0.931 A3A12 0.924 A4B8 0.356 A5A12 0.806 A5B1Q0.915 ASCi 0.966

EXAMPLE 19

RNA Analysis (Northern Blot) of the Human TNF Receptor

1 μg of poly-A⁺ RNA isolated from HS913T fibrosarcoma cells, placenta,and spleen was separated by electrophoresis in a 1.5% vertical agarosegel (10 mM Na phosphate buffer pH=7.0, 6.7% formaldehyde). The sizemarker used was a kilobase ladder radioactively labeled by a fill-inreaction with (α-³²P)dCTP and Klenow enzyme (Bethesda ResearchLaboratories). The formaldehyde was removed from the gel by irrigationand the RNA was transferred in 20×SSC to a nylon membrane (Genescreenplus; NEN-DuPont). The RNA was covalently bonded on the membrane by UVirradiation (100 seconds). The membrane was prehybridized for 2 hours at65° C. in Church buffer (Church and Gilbert, 1984) (0.5 M Na phosphate,pH 7.2, 7% SDS, 1 mM EDTA) and hybridized for 19 hours at 65° C. infresh Church buffer with 3×10⁶ cpm ³²P-labeled DNA probe (EcoRI insertof pTNF-BP30). The filter was washed three times for 10 minutes atambient temperature in washing buffer (40 mM Na phosphate, pH 7.2, 1%SDS) and then four times for 30 minutes at 65° C. in washing buffer, andexposed to Kodak XAR X-ray film for 18 hours using an intensifier screenat −70° C.

The autoradiogram (FIG. 10) shows a singular RNA band with a length of2.3 kb for the human TNF receptor in the analyzed tissues or the cellline HS913T.

EXAMPLE 20

Expression of the TNF Receptor

For transient expression 5–10×10⁷ COS-7 cells were incubated for 40minutes with 10 μg of pADTNF-R plasmid DNA in a solution containing 250μg/ml of DEAE dextran and 50 μg/ml of chloroquin. pADCMV-1-DNA was usedas a control. After transfection, the cells were washed and thencultured for 48 hours. The expression of the TNF receptor wasdemonstrated by ¹²⁵I-TNF binding. For the binding tests, the cells werewashed, incubated for one hour at 4° C. with 10 mg of ¹²⁵I-TNF (specificradioactivity 38,000 cpm/ng) with or without a 200-fold excess ofunlabeled TNF, and the radioactivity bound to the cells was measured ina gamma-counter. The specific binding in the control sample was 2062 cpmand in the samples transformed with TNF receptor DNA it was 6150 cpm;the values are expressed as the average bound cpm; the standarddeviation determined from parallel tests was taken into account. Thenon-specific background in the presence of unlabeled TNF was subtractedfrom the values.

1. A method for ameliorating the harmful effects of TNF in an animal,comprising administering to an animal in need of such treatment atherapeutically effective amount of a homogeneous polypeptide having theability to bind TNF, wherein said polypeptide is encoded by a nucleicacid molecule comprising the nucleotide sequence as set forth in SEQ IDNO:
 3. 2. A method for ameliorating the harmful effects of TNF in ananimal, comprising administering to an animal in need of such treatmenta therapeutically effective amount of a homogeneous polypeptide havingthe ability to bind TNF, wherein said polypeptide comprises the aminoacid sequence as set forth in SEQ ID NO:
 4. 3. A method for amelioratingthe harmful effects of TNF in an animal, comprising administering to ananimal in need of such treatment a therapeutically effective amount of ahomogeneous polypeptide having the ability to bind TNF, wherein saidpolypeptide consists of the amino acid sequence of SEQ ID NO:
 4. 4. Amethod for ameliorating the harmful effects of TNF in an animal,comprising administering to an animal in need of such treatment atherapeutically effective amount of a recombinant polypeptide having theability to bind TNF, wherein said polypeptide is nonglycosylated or isglycosylated by a CHO cell, and wherein said polypeptide consists of theamino acid sequence of SEQ ID NO: 4 and an amino-terminal methionine. 5.A method for ameliorating the harmful effects of TNF in an animal,comprising administering to an animal in need of such treatment atherapeutically effective amount of a polypeptide having the ability tobind TNF, wherein said polypeptide is encoded by a nucleic acid moleculecomprising the nucleotide sequence as set forth in SEQ ID NO: 3, andwherein said polypeptide is expressed from a gene that does not encode:a. amino acid residues 1-29 in SEQ ID NO: 2; b. amino acid residues30-40 in SEQ ID NO: 2; c. amino acid residues 1-40 in SEQ ID NO: 2; ord. amino acid residues 202-455 in SEQ ID NO:
 2. 6. A method forameliorating the harmful effects of TNF in an animal, comprisingadministering to an animal in need of such treatment a therapeuticallyeffective amount of a polypeptide having the ability to bind TNF,wherein said polypeptide comprises the amino acid sequence as set forthin SEQ ID NO: 4, and wherein said polypeptide is expressed from a genethat does not encode: a. amino acid residues 1-29 in SEQ ID NO: 2; b.amino acid residues 30-40 in SEQ ID NO: 2; c. amino acid residues 1-40in SEQ ID NO: 2; or d. amino acid residues 202-455 in SEQ ID NO:
 2. 7. Amethod for ameliorating the harmful effects of TNF in an animal,comprising administering to an animal in need of such treatment atherapeutically effective amount of a polypeptide having the ability tobind TNF, wherein said polypeptide consists of the amino acid sequenceof SEQ ID NO: 4, and wherein said polypeptide is expressed from a genethat does not encode: a. amino acid residues 1-29 in SEQ ID NO: 2; b.amino acid residues 30-40 in SEQ ID NO: 2; c. amino acid residues 1-40in SEQ ID NO: 2; or d. amino acid residues 202-455 in SEQ ID NO:
 2. 8.The method of any of claims 1, 2, 5, or 6 wherein said polypeptide hasat least one additional amino acid at the amino-terminus, at thecarboxyl-terminus, or at both the amino-terminus and thecarboxyl-terminus.
 9. The method of claim 8, wherein said polypeptidehas at least one additional amino acid at the amino-terminus.
 10. Themethod of claim 9, wherein said polypeptide has a methionine at theamino-terminus.
 11. The method of claim 8, wherein said polypeptide hasat least one additional amino acid at the carboxyl-terminus.
 12. Themethod of any of claims 1, 2, 3, 4, 5, 6, or 7, wherein said polypeptideis chemically derivatized.
 13. The polypeptide of any of claims 1, 2, 3,4, 5, 6, or 7, wherein said polypeptide is not glycosylated.
 14. Thepolypeptide of any of claims 1, 2, or 4,wherein said polypeptide isglycosylated.
 15. The polypeptide of claim 14, wherein said polypeptideis glycosylated by a CHO cell.
 16. The method of any of claims 1, 2, 3,4, 5, 6, or 7, wherein said recombinant polypeptide is expressed in acultural cell in vitro and said polypeptide is isolated therefrom. 17.The method of claim 16, wherein the cultured cell is a non-human cell.18. The method of claim 17, wherein the non-human cell is a prokaryoticcell.
 19. The method of claim 18, wherein the prokaryotic cell isEscherichia coli.
 20. The method of claim 17, wherein the non-human cellis a eukaryotic cell.
 21. The method of claim 20, wherein the eukaryoticcell is a mammalian cell.
 22. The method of claim 21, wherein themammalian cell is a Chinese Hamster Ovary cell or a COS cell.
 23. Themethod of claim 16, wherein the polypeptide is glycosylated.
 24. Themethod of claim 16, wherein the polypeptide is not glycosylated.